CN102272306B - The anti-antibody of Siglec 15 - Google Patents

Abstract

A pharmaceutical composition for treating and/or preventing abnormal bone metabolism targeting a protein encoded by a gene strongly expressed in osteoclasts is disclosed. Specifically, a pharmaceutical composition containing an antibody that specifically recognizes human Siglec-15 and has an activity of inhibiting osteoclast formation, etc. is disclosed.

Description

Anti-Siglec-15 antibody

technical field

The present invention relates to a substance useful as a therapeutic and/or preventive agent for abnormal bone metabolism and a method for treating and/or preventing abnormal bone metabolism.

Background technique

Bone is known to be a dynamic organ that undergoes continuous remodeling through repeated formation and resorption, thereby changing its own morphology and maintaining blood calcium levels. Healthy bones maintain a balanced relationship between bone formation by osteoblasts and bone resorption by osteoclasts so that bone mass remains constant. However, when the balance between bone formation and bone resorption is disturbed, abnormalities of bone metabolism such as osteoporosis arise (WO 07/093042 and Endocrinological Review, (1992) 13, pp. 66-80).

As factors regulating bone metabolism, many systemic hormones and local cytokines have been reported, and these factors cooperate with each other to form and maintain bone (WO 07/093042 and Endocrinological Review, (1996) 17, pp. 308-332). The onset of osteoporosis is well known as a change in bone tissue due to aging, but its pathogenesis involves various factors, such as decreased secretion of sex hormones and abnormality of hormone receptors, changes in the expression of cytokines in bone parts, and changes in aging genes. Expression, osteoclast or osteoblast differentiation decline or insufficiency, therefore, it is difficult to understand it as a purely age-related physiological phenomenon. Primary osteoporosis is mainly divided into postmenopausal osteoporosis caused by decreased estrogen secretion and senile osteoporosis caused by aging. In order to elucidate its pathogenesis and develop therapeutic agents against it, in Advances in basic research on the regulatory mechanisms of bone formation and bone resorption are important.

Osteoclasts, which are multinucleated cells derived from hematopoietic stem cells, acidify the space between the bone surface and osteoclasts by releasing chloride and hydrogen ions on the bone surface to which the osteoclasts adhere, and also secrete Cathepsin K etc. which are acidic proteases (American Journal of Physiology, (1991) 260, C1315-C1324). This triggers the degradation of calcium phosphate, the activation of acid proteases, and the degradation of bone matrix proteins, leading to bone resorption.

It has been found that osteoclast precursor cells differentiate into osteoclasts by stimulation of RNAKL (receptor activator of NF-κB ligand) expressed on the cell membrane of osteoblast/stromal cells present on the bone surface (Proceedings of the National Academy of Science of the United States of America, (1998) 95, pp. 3597-3602, and Cell, (1998) 93, pp. 165-176). It has been found that RNAKL is a membrane protein produced by osteoblast/stromal cells, its expression is regulated by bone resorption factors, RNAKL induces the differentiation of osteoclast precursor cells into mature multinucleated osteoclasts, etc. (Proceedings of the National Academy of Science of the United States of America, (1998) 95, pp. 3597-3602, and Journal of Bone and Mineral Research, (1998) 23, S222). In addition, RNAKL knockout mice have been found to develop osteopetrosis-like disease, and thus, RNAKL has been demonstrated to be a physiological osteoclast differentiation inducer (Nature, (1999) 397, pp. 315-323).

Use of bisphosphonates, active vitamin D ₃ , calcitonin and its derivatives, hormones such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K ₂ (menadione ), PTH and calcium preparations are used as pharmaceutical preparations for treating bone metabolic diseases or shortening the treatment time. However, these drugs do not always exhibit satisfactory therapeutic effects, and development of drugs with more effective therapeutic effects has been demanded.

The cell membranes of immune cells are highly covered with glycans, such as sialylated glycans, which are recognized by various glycan-binding proteins. Sialic acid-binding immunoglobulin-like lectins (hereinafter "Siglecs") are a family of type I membrane proteins that recognize and bind sialylated glycans. Many siglecs are expressed on the cell membrane of immune cells and recognize sialic acid similarly present on the cell membrane of immune cells, regulate cell interactions or cell functions, and are thought to be involved in immune responses (Nature Reviews Immunology, (2007) 7, p. 255-266). However, there are also many siglec molecules whose physiological functions have not yet been elucidated. Siglec-15 (sialic acid-binding immunoglobulin-like lectin 15) was recently reported as a molecule belonging to Siglecs (see, for example, Non-Patent Document 10), which is identical to the molecule called CD33L3 (CD33 molecule-like 3). This molecule is highly evolutionarily conserved from fish to humans and has been found to be strongly expressed in dendritic cells and/or macrophages of human spleen and lymph nodes. Furthermore, as a result of a binding test using a sialic acid probe, it has been found that human Siglec-15 binds to Neu5Acα2-6GalNAc, and mouse Siglec-15 further binds to Neu5Acα2-3Galβ1-4Glc, etc. (see, for example, Glycobiology, (2007) 17, pp. 838-846). Until recently, the physiological role of Siglec-15 has not been revealed, but it has been reported that the expression of Siglec-15 increases with the differentiation and maturation of osteoclasts, and osteoclasts can be inhibited by reducing the expression of Siglec-15 by RNA interference Differentiation of cells (see, eg, WO 07/093042). Furthermore, the effect of anti-Siglec-15 antibody on osteoclast differentiation has been disclosed for the first time in WO 09/48072 (published April 16, 2009), however, the antibody sequence that can be administered to humans has not been elucidated.

Contents of the invention

The problem to be solved by the invention

The object of the present invention is to provide: genes specifically expressed in various forms of abnormal bone metabolism (as seen in osteoporosis, rheumatoid arthritis, bone metastases of cancer, etc.); inhibition of osteoclast differentiation and maturation; active substances thereof; and therapeutic and/or preventive agents for abnormal bone metabolism.

means of solving problems

In order to find a substance having a therapeutic and/or preventive effect on abnormal bone metabolism, the present inventors conducted research with the aim of elucidating the mechanism of differentiation, maturation and activation of osteoclasts. As a result, the present inventors found that the expression of Siglec-15 gene increases with the differentiation and maturation of osteoclasts, and also found that an antibody specifically binding to Siglec-15 can inhibit the differentiation of osteoclasts. Furthermore, the inventors humanized the obtained rat anti-mouse Siglec-15 antibody to complete the present invention.

That is, the present invention includes the following inventions.

(1) An antibody or a functional fragment of the antibody that binds to a polypeptide comprising amino acid residues 39-165 of the amino acid sequence represented by SEQ ID NO: 2 and inhibits osteoclastogenesis and/or osteoclastosis Sexual bone resorption.

(2) The antibody or the functional fragment of the antibody according to (1), characterized in that:

The heavy chain sequence contains a variable region having CDRH1, CDRH2 and CDRH3, and the CDRH1 comprises the amino acid sequence represented by SEQ ID NO: 44, and the CDRH2 comprises any one of SEQ ID NO: 45 and SEQ ID NO: 97 an amino acid sequence, said CDRH3 comprising the amino acid sequence represented by SEQ ID NO: 46; and

The light chain sequence comprises a variable region having CDRL1, CDRL2 and CDRL3, and said CDRL1 comprises an amino acid sequence represented by SEQ ID NO: 47, said CDRL2 comprises an amino acid sequence represented by SEQ ID NO: 48, and said CDRL3 comprises Amino acid sequence represented by SEQ ID NO: 49.

(3) The antibody or the functional fragment of the antibody according to (2), characterized by comprising: a heavy chain variable region sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 41 and comprising The light chain variable region sequence of amino acid residues 21-132 of the amino acid sequence represented by SEQ ID NO: 43.

(4) The antibody functional fragment according to any one of (1) to (3), which is selected from the group consisting of: Fab, F(ab')2, Fab' and Fv.

(5) The antibody according to any one of (1) to (3), which is an scFv.

(6) The antibody or the functional fragment of the antibody according to any one of (1) to (4), characterized in that the antibody is a chimeric antibody.

(7) The antibody or the functional fragment of the antibody according to (6), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 41 bases 20-470, and the light chain sequence comprises amino acid residues 21-237 of the amino acid sequence represented by SEQ ID NO: 43.

(8) The antibody or the functional fragment of the antibody according to any one of (1) to (4) and (6), characterized in that the antibody is humanized.

(9) The antibody according to (7) or (8), wherein the heavy chain has a constant region of a human immunoglobulin G2 heavy chain, and the light chain has a constant region of a human immunoglobulin κ light chain.

(10) An antibody or a functional fragment of the antibody that inhibits osteoclast formation and/or osteoclastic bone resorption, wherein the antibody contains:

(a) a heavy chain variable region selected from the following amino acid sequences:

a1) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 51;

a2) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 53;

a3) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 55;

a4) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 57;

a5) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 59;

a6) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 99;

a7) an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 101;

a8) an amino acid sequence having at least 95% homology to any one of the amino acid sequences selected from a1) to a7);

a9) an amino acid sequence having at least 99% homology to any one of the amino acid sequences selected from a1) to a7); and

a10) an amino acid sequence comprising a substitution, deletion or addition of one to several amino acid residues in any one of the amino acid sequences selected from a1) to a7); and

(b) a light chain variable region selected from the following amino acid sequences:

b1) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 61;

b2) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 63;

b3) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 65;

b4) an amino acid sequence comprising amino acid residues 21-132 of the amino acid sequence represented by SEQ ID NO: 67;

b5) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 69;

b6) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 71;

b7) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 103;

b8) an amino acid sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 105;

b9) an amino acid sequence having at least 95% homology to any one of the amino acid sequences selected from b1) to b8);

b10) an amino acid sequence having at least 99% homology to any one of the amino acid sequences selected from b1) to b8); and

b11) An amino acid sequence comprising a substitution, deletion or addition of one to several amino acid residues in any one of the amino acid sequences selected from b1) to b8).

(11) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 51, and the light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 61.

(12) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 53, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 63.

(13) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 55, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 65.

(14) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 55, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-132 of the amino acid sequence represented by SEQ ID NO: 67.

(15) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 57, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 69.

(16) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 59, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 71.

(17) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 99, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 69.

(18) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 101, and the light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 69.

(19) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 99, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 103.

(20) The antibody or the functional fragment of the antibody according to (10), characterized in that it contains a heavy chain sequence and a light chain sequence, the heavy chain sequence contains a heavy chain variable region, and the heavy chain variable region comprising an amino acid sequence comprising amino acid residues 20-140 of the amino acid sequence represented by SEQ ID NO: 99, and said light chain sequence comprising a light chain variable region comprising such amino acids A sequence comprising amino acid residues 21-133 of the amino acid sequence represented by SEQ ID NO: 105.

(21) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 51 bases 20-470, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 61.

(22) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 53 bases 20-470, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 63.

(23) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 55 bases 20-470, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 65.

(24) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 55 bases 20-470, and the light chain sequence comprises amino acid residues 21-237 of the amino acid sequence represented by SEQ ID NO: 67.

(25) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 57 bases 20-470, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 69.

(26) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 59 bases 20-470, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 71.

(27) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 99 bases 20-466, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 69.

(28) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 101 bases 20-466, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 69.

(29) The antibody or the functional fragment of the antibody according to (10), characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 99 bases 20-466, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 103.

(30) The antibody according to (10) or the functional fragment of the antibody, characterized by comprising a heavy chain sequence and a light chain sequence, the heavy chain sequence comprising amino acid residues of the amino acid sequence represented by SEQ ID NO: 99 bases 20-466, and the light chain sequence comprises amino acid residues 21-238 of the amino acid sequence represented by SEQ ID NO: 105.

(31) A pharmaceutical composition characterized by comprising at least one antibody or a functional fragment of the antibody according to (1) to (30).

(32) The pharmaceutical composition according to (31), which is a therapeutic and/or preventive agent for abnormal bone metabolism.

(33) A pharmaceutical composition for treating and/or preventing abnormal bone metabolism, characterized by comprising at least one antibody or functional fragment of the antibody according to (1) to (30) and at least one selected Members from: bisphosphonates, active vitamin D ₃ , calcitonin and its derivatives, hormones such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K ₂ ( Menadione), calcium preparations, PTH (parathyroid hormone), nonsteroidal anti-inflammatory drugs, soluble TNF receptors, anti-TNF-α antibodies or functional fragments of said antibodies, anti-PTHrP (thyroid parathyroid hormone-related protein) antibody or a functional fragment of the antibody, an IL-1 receptor antagonist, an anti-IL-6 receptor antibody or a functional fragment of the antibody, an anti-RANKL antibody or a functional fragment of the antibody and OCIF (Osteoclastogenesis Inhibitory Factor).

(34) The pharmaceutical composition according to (32) or (33), wherein the abnormality in bone metabolism is selected from the group consisting of osteoporosis, bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, Bone destruction in bone metastases of multiple myeloma or carcinoma, giant cell tumor, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, Renal osteodystrophy and osteogenesis imperfecta.

(35) The pharmaceutical composition according to (34), wherein the abnormal bone metabolism is osteoporosis, bone destruction associated with rheumatoid arthritis, or bone destruction associated with bone metastasis of cancer.

(36) The pharmaceutical composition according to (35), characterized in that the abnormal bone metabolism is osteoporosis.

(37) The pharmaceutical composition according to (36), characterized in that the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, caused by the use of therapeutic agents such as steroids or immunosuppressants. secondary osteoporosis, or osteoporosis associated with rheumatoid arthritis.

(38) A method for treating and/or preventing abnormal bone metabolism, characterized by administering at least one antibody or a functional fragment of the antibody according to (1) to (30).

(39) A method for treating and/or preventing abnormal bone metabolism, characterized in that at least one antibody or functional fragment of the antibody according to (1) to (30) and at least one selected from Members of the following: bisphosphonates, active vitamin _D3 , calcitonin and its derivatives, hormones such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K2 ( _four menadione), calcium preparations, PTH (parathyroid hormone), nonsteroidal anti-inflammatory drugs, soluble TNF receptors, anti-TNF-α antibodies or functional fragments of said antibodies, anti-PTHrP (parathyroid GRP) antibody or a functional fragment of said antibody, an IL-1 receptor antagonist, an anti-IL-6 receptor antibody or a functional fragment of said antibody, an anti-RANKL antibody or a functional fragment of said antibody, and OCIF (Osteoclastogenesis Inhibitory Factor).

(40) The treatment and/or prevention method according to (38) or (39), wherein the bone metabolism abnormality is osteoporosis, bone destruction accompanied by rheumatoid arthritis, or bone disease accompanied by cancer. Metastatic bone destruction.

(41) The treatment and/or prevention method according to (40), characterized in that the abnormal bone metabolism is osteoporosis.

(42) The method of treatment and/or prevention according to (41), characterized in that the osteoporosis is postmenopausal osteoporosis, senile osteoporosis, caused by the use of such as steroids or immunosuppressants, etc. Osteoporosis secondary to therapeutic agents, or osteoporosis associated with rheumatoid arthritis.

(43) A polynucleotide encoding the antibody according to any one of (3), (7) and (10) to (30).

(44) The polynucleotide according to (43), characterized by comprising: a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 40, and comprising the nucleotide sequence represented by SEQ ID NO: 40 The nucleotide sequence of nucleotides 61-396 of the nucleotide sequence represented by NO: 42.

(45) The polynucleotide according to (44), characterized by comprising: a nucleotide sequence comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 40, and comprising the nucleotide sequence represented by SEQ ID NO: 40 The nucleotide sequence of nucleotides 61-711 of the nucleotide sequence represented by NO: 42.

(46) The polynucleotide according to (43), comprising:

(a) a polynucleotide selected from the following nucleotide sequences:

a1) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 50;

a2) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 52;

a3) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 54;

a4) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 56;

a5) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 58;

a6) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 98;

a7) a nucleotide sequence comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 100;

a8) a nucleotide sequence of a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a complementary nucleotide sequence to any one of the nucleotide sequences selected from a1) to a7); and

a9) a nucleotide sequence comprising a substitution, deletion or addition of one to several nucleotides in any one of the nucleotide sequences selected from a1) to a7); and

(b) a polynucleotide selected from the following nucleotide sequences:

b1) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 60;

b2) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 62;

b3) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 64;

b4) a nucleotide sequence comprising nucleotides 61-396 of the nucleotide sequence represented by SEQ ID NO: 66;

b5) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 68;

b6) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 70;

b7) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 102;

b8) a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 104;

b9) a nucleotide sequence of a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising a complementary nucleotide sequence to any one of the nucleotide sequences selected from b1) to b8); and

b10) A nucleotide sequence comprising a substitution, deletion or addition of one to several nucleotides in any one of the nucleotide sequences selected from b1) to b8).

(47) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 50 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 60.

(48) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 52 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 62.

(49) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 54 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 64.

(50) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 54 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-396 of the nucleotide sequence represented by SEQ ID NO: 66.

(51) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 56 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 68.

(52) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 58 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO:70.

(53) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 68.

(54) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58 to 420 of the nucleotide sequence represented by SEQ ID NO: 100 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 68.

(55) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 102.

(56) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-420 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-399 of the nucleotide sequence represented by SEQ ID NO: 104.

(57) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 50 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 60.

(58) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 52 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 62.

(59) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 54 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 64.

(60) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 54 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-711 of the nucleotide sequence represented by SEQ ID NO: 66.

(61) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 56 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 68.

(62) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1410 of the nucleotide sequence represented by SEQ ID NO: 58 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO:70.

(63) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1398 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 68.

(64) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1398 of the nucleotide sequence represented by SEQ ID NO: 100 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 68.

(65) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1398 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 102.

(66) The polynucleotide according to (46), characterized by comprising: a polynucleotide comprising nucleosides comprising nucleotides 58-1398 of the nucleotide sequence represented by SEQ ID NO: 98 acid sequence; and a polynucleotide comprising a nucleotide sequence comprising nucleotides 61-714 of the nucleotide sequence represented by SEQ ID NO: 104.

(67) A vector comprising any one of the polynucleotides according to (43) to (66).

(68) A transformed host cell comprising the polynucleotide according to any one of (43) to (66).

(69) A transformed host cell comprising the vector according to (67).

(70) A method of producing the antibody according to any one of (3), (7) and (10) to (30), the method comprising: culturing the antibody according to (68) or (69) host cells, and antibodies were purified from the resulting culture products.

Advantages of the invention

According to the present invention, a therapeutic and/or preventive agent for abnormal bone metabolism can be obtained, the mechanism of action of which is to inhibit the differentiation and maturation of osteoclasts and their bone resorption activity.

Description of drawings

Figure 1 shows the results of evaluating the purity of mouse Siglec-15-His purified by HisTrap HP column chromatography and Resource Q column chromatography by SDS-polyacrylamide electrophoresis and silver staining.

Fig. 2 shows the results of detecting the characteristics of mouse Siglec-15-His purified by HisTrap HP column chromatography and Resource Q column chromatography by SDS-polyacrylamide electrophoresis and Western blotting using anti-V5-HRP antibody.

Fig. 3 shows the results of evaluating the purity of mouse Siglec-15-Fc purified by HiTrap protein A column chromatography by SDS-polyacrylamide electrophoresis and silver staining.

Fig. 4 shows the results of testing the binding of rat anti-mouse Siglec-15 monoclonal antibody to a plate on which mouse Siglec-15-Fc was immobilized by the ELISA method. The symbol (◆) represents #1A1 antibody, the symbol (■) represents #3A1 antibody, the symbol (▲) represents #8A1 antibody, the symbol (×) represents #24A1 antibody, the symbol (●) represents #32A1 antibody, and the symbol (○) represents #34A1 antibody, symbol (+) indicates #39A1 antibody, symbol (-) indicates #40A1 antibody, symbol (-) indicates #41B1 antibody, symbol (◇) indicates #61A1 antibody, and symbol (□) indicates control IgG.

Figure 5 shows the results of testing the effect of adding rat anti-mouse Siglec-15 monoclonal antibody (#3A1, #8A1 or #32A1) on the differentiation of mouse bone marrow non-adherent cells into osteoclasts (stimulated with RANKL) . In addition, the rat control IgG in the figure is a common negative control in Figures 5 and 6.

Figure 6 shows the results of testing the effect of adding rat anti-mouse Siglec-15 monoclonal antibody (#34A1, #39A1 or #40A1) on the differentiation of mouse bone marrow non-adherent cells into osteoclasts (stimulated with RANKL) . In addition, rabbit anti-mouse Siglec-15 polyclonal antibody No. 3 in the figure is a common positive control in FIGS. 5 and 6 .

Fig. 7 shows the results of evaluating the purity of human Siglec-15-His purified by HisTrap HP column chromatography and Resource Q column chromatography by SDS-polyacrylamide electrophoresis and silver staining.

Fig. 8 shows the results of evaluating the purity of human Siglec-15-Fc purified by protein A column chromatography by SDS-polyacrylamide electrophoresis.

Fig. 9 shows the results of testing the binding of rat anti-mouse Siglec-15 monoclonal antibody to the plate on which human Siglec-15-Fc was immobilized by the ELISA method. The symbol (◆) represents #1A1 antibody, the symbol (■) represents #3A1 antibody, the symbol (▲) represents #8A1 antibody, the symbol (×) represents #24A1 antibody, the symbol (●) represents #32A1 antibody, and the symbol (○) represents #34A1 antibody, symbol (+) indicates #39A1 antibody, symbol (-) indicates #40A1 antibody, symbol (-) indicates #41B1 antibody, symbol (◇) indicates #61A1 antibody, and symbol (□) indicates control IgG.

Figure 10 shows photomicrographs of inhibition of giant osteoclast formation from normal human osteoclast precursor cells by addition of rat anti-mouse Siglec-15 monoclonal antibody depicted by TRAP staining.

Figure 11 shows photomicrographs of inhibition of giant osteoclast formation from normal human osteoclast precursor cells by addition of rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) depicted by TRAP staining.

Fig. 12 is a graph showing inhibition of bone resorption activity of normal human osteoclasts by adding rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) (N=6).

Figure 13A is a graph showing the effect of increasing lumbar bone mineral density when rat anti-mouse Siglec-15 monoclonal antibody was administered to ovariectomized rats for 4 weeks; Effect of mouse anti-mouse Siglec-15 monoclonal antibody on reducing urinary deoxypyridinoline excretion when administered to ovariectomized rats for 4 weeks.

Figure 14 is a graph showing that rat anti-mouse Siglec-15 monoclonal antibody #32A1 binds to the V-group domain of human Siglec-15 by competition ELISA.

Figure 15 is a graph showing that rat #32A1 antibody and its human chimeric antibody have substantially the same affinity for mouse Siglec-15-Fc by competition ELISA.

Figure 16 shows a graph of the inhibition of mouse osteoclast formation by the addition of rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) and its chimeric antibody as depicted by TRAP enzyme activity (N=3).

Figure 17 shows photomicrographs of inhibition of giant osteoclast formation from normal human osteoclast precursor cells by addition of human chimeric antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1 depicted by TRAP staining.

Fig. 18 is a graph in which it was confirmed by the ELISA method using a plate on which mouse Siglec-15-Fc was immobilized that 6 types of humanized rat anti-mouse Siglec-15 antibodies exhibited an antibody concentration-dependent Binding to mouse Siglec-15 protein.

Fig. 19 is a graph in which it was confirmed that six types of humanized rat anti-mouse Siglec-15 antibodies bind in an antibody concentration-dependent manner by the ELISA method using a plate on which human Siglec-15-Fc is immobilized Human Siglec-15 protein.

Fig. 20 shows photomicrographs of inhibition of formation of mouse giant osteoclasts under TNFα stimulation by addition of rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) or its chimeric antibody depicted by TRAP staining.

Figure 21 shows the graph of inhibition of formation of mouse osteoclasts under TNFα stimulation by adding rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) or its chimeric antibody, depicted by TRAP enzyme activity (N= 3).

Figure 22 shows a graph of inhibition of rat osteoclast formation by addition of rat anti-mouse Siglec-15 monoclonal antibody (#8A1 or #32A1 antibody) plotted with TRAP enzyme activity (N=3).

Figure 23 shows the nucleotide sequence of the cloned rat #32A1 heavy chain and its amino acid sequence.

Figure 24 shows the nucleotide sequence of the cloned rat #32A1 light chain and its amino acid sequence.

Figure 25 shows the nucleotide sequence and amino acid sequence of #32A1 human chimeric antibody heavy chain.

Figure 26 shows the nucleotide sequence and amino acid sequence of #32A1 human chimeric antibody light chain.

Figure 27 shows the nucleotide sequence of h#32A1-T1H and its amino acid sequence.

Fig. 28 shows the nucleotide sequence of h#32A1-T2H and its amino acid sequence.

Fig. 29 shows the nucleotide sequence of h#32A1-T3H and its amino acid sequence.

Figure 30 shows the nucleotide sequence of h#32A1-T5H and its amino acid sequence.

Figure 31 shows the nucleotide sequence of h#32A1-T6H and its amino acid sequence.

Figure 32 shows the nucleotide sequence of h#32A1-T1L and its amino acid sequence.

Figure 33 shows the nucleotide sequence of h#32A1-T2L and its amino acid sequence.

Figure 34 shows the nucleotide sequence of h#32A1-T3L and its amino acid sequence.

Figure 35 shows the nucleotide sequence of h#32A1-T4L and its amino acid sequence.

Figure 36 shows the nucleotide sequence of h#32A1-T5L and its amino acid sequence.

Figure 37 shows the nucleotide sequence of h#32A1-T6L and its amino acid sequence.

Figure 38 shows the amino acid sequences of h#32A1-T7H, h#32A1-T8H and h#32A1-T9H.

Figure 39 shows the amino acid sequences of h#32A1-T10H, h#32A1-T11H and h#32A1-T12H.

Figure 40 shows the amino acid sequences of h#32A1-T7L, h#32A1-T8L, h#32A1-T9L, h#32A1-T10L and h#32A1-T11L.

Figure 41 shows the amino acid sequences of h#32A1-T12L, h#32A1-T13L, h#32A1-T14L, h#32A1-T15L and h#32A1-T16L.

Figure 42 shows the amino acid sequences of h#32A1-T17L, h#32A1-T18L, h#32A1-T19L, h#32A1-T20L and h#32A1-T21L.

Figure 43 shows the amino acid sequences of h#32A1-T22L, h#32A1-T23L, h#32A1-T24L and h#32A1-T25L.

Figure 44 shows inhibition of mouse osteoclastosis by addition of humanized rat anti-mouse Siglec-15 antibody (h#32A1-T1, h#32A1-T2 or h#32A1-T3) as depicted by TRAP enzyme activity Diagram of cell formation. In addition, the rat #32A1 antibody in the figure is a positive control common to both Figures 44 and 45.

Figure 45 shows inhibition of mouse osteoclastosis by addition of humanized rat anti-mouse Siglec-15 antibody (h#32A1-T4, h#32A1-T5 or h#32A1-T6) as depicted by TRAP enzyme activity Diagram of cell formation.

Figure 46 shows inhibition of mouse osteoclastosis by addition of humanized rat anti-mouse Siglec-15 antibody (h#32A1-H1-1/L5 or h#32A1-H5/L5) as depicted by TRAP enzyme activity Diagram of cell formation. In addition, rat #32A1 antibody and #32A1 human chimeric antibody in the figure are positive controls common to Figures 46 and 47.

Figure 47 shows the activity profiled by TRAP by adding humanized rat anti-mouse Siglec-15 antibody (h#32A1-H1-1/L2-16 or h#32A1-H-1/L2-15 ) to inhibit the formation of mouse osteoclasts.

Figure 48 shows a graph depicting the addition of humanized rat anti-mouse Siglec-15 antibodies (h#32A1-T1, h#32A1-T2, h#32A1-T3, h#32A1-T4, h# 32A1-T5 or h#32A1-T6) inhibit the bone resorption activity of normal human osteoclasts. In addition, rat #32A1 antibody and #32A1 human chimeric antibody in the figure are positive controls common to Figures 48 and 49 .

Figure 49 shows a graph depicting the addition of humanized rat anti-mouse Siglec-15 antibodies (h#32A1-H1-1/L5, h#32A1-H5/L5, h#32A1-H1-1/ L2-16 or h#32A1-H1-1/L2-15) inhibit the bone resorption activity of normal human osteoclasts.

Fig. 50 is a thermogram obtained for measuring the thermal stability of h#32A1-H5/L5 antibody.

Fig. 51 is a thermogram obtained for measuring the thermal stability of h#32A1-H1-1/L5 antibody.

Fig. 52 is a thermogram obtained for measuring the thermal stability of h#32A1-H1-1/L2-15 antibody.

Fig. 53 is a thermogram obtained for measuring the thermal stability of h#32A1-H1-1/L2-16 antibody.

Figure 54 shows the nucleotide sequence of h#32A1-H1-1 and its amino acid sequence.

Figure 55 shows the nucleotide sequence of h#32A1-H5 and its amino acid sequence.

Figure 56 shows the nucleotide sequence of h#32A1-L2-15 and its amino acid sequence.

Figure 57 shows the nucleotide sequence of h#32A1-L2-16 and its amino acid sequence.

detailed description

The term "gene" used herein includes not only DNA but also mRNA, cDNA and cRNA.

As used herein, the term "polynucleotide" is used in the same sense as nucleic acid, and also includes DNA, RNA, probes, oligonucleotides, and primers.

As used herein, the terms "polypeptide" and "protein" are used without distinction.

The term "RNA fraction" as used herein refers to a fraction comprising RNA.

As used herein, the term "cell" also includes cells in an animal subject and cells in culture.

The term "Siglec-15" used herein is used in the same meaning as "Siglec-15 protein".

As used herein, the term "osteoclast formation" is used in the same sense as "osteoclast differentiation" or "osteoclast maturation".

As used herein, the term "antibody functional fragment" refers to a fragment of an antibody that has antigen-binding activity, including Fab, F(ab')2, Fv, scFv, diabodies, linear antibodies, multispecific antibodies formed from antibody fragments Antibodies, etc. The term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treatment of F(ab')2 under reducing conditions. However, the term is not limited to these molecules so long as the fragment has binding affinity for the antigen. In addition, these functional fragments include not only fragments obtained by treating full-length molecules of antibody proteins with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes.

The term "epitope" as used herein refers to a partial peptide or a partial tertiary structure of Siglec-15 to which a specific anti-Siglec-15 antibody binds. The above-mentioned epitope as a partial peptide of Siglec-15 can be determined by a method well known to those skilled in the art such as immunoassay. However, for example, the following methods may be employed. Preparation of various partial structures of Siglec-15. In preparing partial structures, known oligopeptide synthesis techniques can be used. For example, using gene recombination techniques known to those skilled in the art, a series of polypeptides with appropriate shortened lengths obtained by sequentially shortening the C-terminus or N-terminus of Siglec-15 can be prepared. Thereafter, the reactivity of antibodies to these polypeptides was tested, and the recognition site was roughly determined. Epitopes can then be determined by synthesizing shorter length peptides and testing reactivity with these peptides. Furthermore, by describing the amino acid residues of Siglec-15 adjacent to the antibody by X-ray structural analysis, the epitope (which binds a specific Siglec-15 antibody) that is part of the tertiary structure of Siglec-15 can be determined. If the second anti-Siglec-15 antibody binds to the partial peptide or partial tertiary structure to which the first anti-Siglec-15 antibody binds, it can be determined that the first antibody and the second antibody have the same epitope. In addition, the primary antibody can be determined by demonstrating that the secondary anti-Siglec-15 antibody competes with the primary anti-Siglec-15 antibody for Siglec-15 binding (i.e., the secondary antibody inhibits the binding between Siglec-15 and the primary antibody). have the same epitope as the secondary antibody, even if the sequence or structure of the specific epitope has not been determined. Furthermore, when the first antibody and the second antibody bind to the same epitope, and the first antibody has a specific effect such as antigen neutralizing activity, the second antibody can be expected to have the same activity.

It is known that each heavy and light chain of an antibody molecule has three complementarity determining regions (CDRs). Complementarity determining regions are also called hypervariable domains and are present in the variable region of each heavy and light chain of antibodies. It is a site of very high variability in its primary structure and there are 3 separate CDRs in the primary structure of each heavy and light polypeptide chain. In this specification, regarding the complementarity determining regions of antibodies, the complementarity determining regions of the heavy chain are represented by CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the complementarity determining regions of the light chain are represented by CDRH1, CDRH2, and CDRH3 derived from the amino acid sequence of the light chain. CDRL1, CDRL2, and CDRL3 on the amino-terminal side of . The proximity of these sites to each other in the tertiary structure determines the specificity of the antigen to which the antibody binds.

The phrase "hybridize under stringent conditions" as used herein refers to hybridization under conditions that allow identification, wherein hybridization is performed at 68°C in a commercially available hybridization solution, ExpressHyb hybridization solution (manufactured by Clontech, Inc. , or using a filter with DNA immobilized on it, hybridize at 68°C in the presence of 0.7-1.0M NaCl, and then use a 0.1-2X SSC solution (1 x SSC solution consists of 150 mM NaCl and 15 mM sodium citrate ) were washed at 68°C, or hybridized under the same conditions.

1. Siglec-15

The present inventors have found that the Siglec-15 gene is specifically expressed in giant cell tumors and have also found that the expression level of the Siglec-15 gene increases when monocyte-derived cell lines differentiate into osteoclasts.

Regarding Siglec-15 used in the present invention, Siglec-15 is obtained directly from mononuclear cells of humans, non-human mammals (such as guinea pigs, rats, mice, rabbits, pigs, sheep, cows, or monkeys) or chickens. Or bone marrow cells are purified and used, or cell membrane fractions of the above cells are prepared and used. In addition, Siglec-15 can be obtained by synthesizing Siglec-15 in vitro or producing it in host cells by genetic engineering. Specifically, in the production of genetic engineering, the Siglec-15 cDNA is integrated into a vector capable of expressing the Siglec-15 cDNA, and then Siglec-15 is synthesized in a solution containing enzymes, substrates and energy substances required for transcription and translation, Alternatively, the protein can be obtained by transforming another prokaryotic or eukaryotic host cell to express Siglec-15.

The nucleotide sequence of human Siglec-15 cDNA is registered in GenBank with accession number NM_213602, and is represented as SEQ ID NO: 1 of the sequence listing, and its amino acid sequence is represented as SEQ ID NO: 2 of the sequence listing. The nucleotide sequence of mouse Siglec-15 cDNA is registered in GenBank with the accession number XM_884636, and is represented as SEQ ID NO:3 of the sequence listing, and its amino acid sequence is represented as SEQ ID NO:4 of the sequence listing. Mature human Siglec-15 from which the signal sequence has been removed corresponds to an amino acid sequence comprising amino acid residues 21-328 of the amino acid sequence represented by SEQ ID NO:2. In addition, mouse Siglec-15 from which the signal sequence has been removed corresponds to an amino acid sequence comprising amino acid residues 21-341 of the amino acid sequence represented by SEQ ID NO:4. Additionally, Siglec-15 is sometimes referred to as CD33 antigen-like 3, CD33 molecule-like 3, CD33-like 3, or CD33L3, which all refer to the same molecule.

Siglec-15 cDNA can be obtained by, for example, a so-called PCR method in which a polymerase chain reaction (hereinafter referred to as "PCR") (Saiki, R. K., et al., Science, (1988) 239, 487-49).

In addition, a polynucleotide having the following characteristics is also regarded as Siglec-15 cDNA: it is combined under stringent conditions with at least one complementary nucleoside selected from the nucleotide sequence represented by SEQ ID NO: 1 and 3 in the Sequence Listing The polynucleotides of acid sequence hybridize and encode a protein with biological activity comparable to Siglec-15. In addition, a polynucleotide having the following characteristics is also considered a Siglec-15 cDNA: it is a splice variant transcribed from the human or mouse Siglec-15 locus, or hybridizes under stringent conditions to its complementary sequence, and encodes a Siglec-15 comparable biologically active protein.

In addition, a protein having the following characteristics is also regarded as Siglec-15: it comprises an amino acid sequence comprising one of at least one amino acid sequence selected from the amino acid sequences represented by SEQ ID NO: 2 and 4 of the Sequence Listing Or the substitution, deletion or addition of several amino acids, or the amino acid sequence obtained by removing the signal sequence from any of these sequences, and has a biological activity comparable to that of Siglec-15. In addition, a protein is considered Siglec-15 if it contains an amino acid sequence encoded by a splice variant transcribed from the human or mouse Siglec-15 locus, or a substitution of one or several amino acids contained therein , deleted or added amino acid sequence, and has biological activity comparable to Siglec-15.

2. Detection of Abnormal Bone Metabolism

Analysis of the expression levels of the Siglec-15 gene in test sample sets from various human bone tissues revealed a significant increase in expression levels of this gene in giant cell tumors (GCTs), which are osteoclast-like multinucleated giant cells Bone tumors that occur in large numbers and are characterized clinically by osteolytic bone destruction (Bullough et al., Atlas of Orthopedic Pathology 2nd Ed., pp. 17.6-17.8, Lippincott Williams & Wilkins Publishers (1992)).

It was also found that the expression level of the Siglec-15 gene was increased when monocyte-derived cell lines were differentiated into osteoclasts.

Therefore, Siglec-15 is believed to be involved in human pathologies such as GCT in which bone resorption is increased. In other words, by measuring the Siglec-15 gene and/or the expression level of Siglec-15 in each cell and/or each tissue, the state of abnormal bone metabolism accompanied by overexpression of Siglec-15 can be determined. Abnormal bone metabolism as used herein refers to disorders characterized by net bone loss, specific examples of which include, but are not limited to: osteoporosis (postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis caused by therapeutic agents such as anti-inflammatory drugs, or osteoporosis associated with rheumatoid arthritis), bone destruction associated with rheumatoid arthritis, cancerous hypercalcemia, and multiple myeloma bone metastases of cancer or cancer, giant cell tumor, osteopenia, tooth loss due to periodontitis, osteolysis around artificial joints, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteotrophy Dysfunction and Osteogenesis Imperfecta.

In the present invention, the "test sample" used to detect the Siglec-15 gene and/or the expression level of Siglec-15 refers to samples of the following tissues obtained from test subjects: bone marrow, bone, prostate, testis, penis , bladder, kidneys, oral cavity, pharynx, lips, tongue, gums, nasopharynx, esophagus, stomach, small intestine, large intestine, colon, liver, gallbladder, pancreas, nose, lungs, soft tissue, skin, breast, uterus, ovary, brain , thyroid, lymph node, muscle, adipose tissue, etc., or blood, body fluid, excrement, etc. obtained from test subjects, clinical samples, etc., however, in the present invention, blood or bone marrow is more preferred.

Regarding RANKL, which is known to be involved in osteoclast differentiation, knockout mice have been generated and the phenotype when RANKL function has been lost has been analyzed (Young-Yun Kong, et al., Nature (1999) 397, p. pp. 315-323). By producing a knockout mouse lacking Siglec-15 in the same manner as above, the phenotype when Siglec-15 function has been lost can be analyzed.

3. Preparation of Anti-Siglec-15 Antibody

Anti-Siglec-15 antibodies of the present invention can be obtained by immunizing animals with Siglec-15 or any polypeptide selected from the amino acid sequence of Siglec-15 according to conventional methods, and collecting and purifying the antibodies produced in vivo. The biological species of Siglec-15 used as an antigen is not limited to humans, and animals can be immunized with Siglec-15 obtained from animals other than humans such as mice or rats. In this case, antibodies applicable to human diseases can be selected by testing the cross-reactivity of the antibodies with the resulting binding of heterologous Siglec-15 and human Siglec-15.

Furthermore, according to known methods (for example, Kohler and Milstein, Nature, (1975) 256, pp. 495-497; Kennet, R. ed., Monoclonal Antibody, pp. 365-367, Prenum Press, N.Y. (1980)) , Monoclonal antibodies can be obtained by fusing antibody-producing cells that produce anti-Siglec-15 antibodies with myeloma cells to establish hybridomas.

In addition, Siglec-15 to be used as an antigen can be obtained by causing a host cell to produce the Siglec-15 gene by genetic engineering.

Specifically, a vector capable of expressing the Siglec-15 gene is prepared, the obtained vector is transfected into a host cell to express the gene, and then the expressed Siglec-15 is purified. The methods for obtaining anti-Siglec-15 antibodies are specifically described below.

(1) Antigen preparation

Examples of antigens for preparing anti-Siglec-15 antibodies include Siglec-15, a polypeptide comprising a partial amino acid sequence containing at least 6 consecutive amino acids of Siglec-15, and a polypeptide obtained by adding a given amino acid sequence or a vector thereto Derivatives.

Siglec-15 can be directly purified from human tumor tissue or tumor cells and used. In addition, Siglec-15 can be obtained by in vitro synthesis or by causing host cells to produce Siglec-15 by genetic engineering.

Regarding genetic engineering, specifically, Siglec-15 cDNA is integrated into a vector capable of expressing Siglec-15 cDNA, and then Siglec-15 is synthesized in a solution containing enzymes, substrates and energy substances required for transcription and translation, or transformed Another prokaryotic or eukaryotic host cell to express Siglec-15 from which the antigen can be obtained.

In addition, by expressing a fusion protein obtained by linking the extracellular domain of Siglec-15 as a membrane protein to the constant region of an antibody in an appropriate host-vector system, it is also possible to obtain antigen.

Siglec-15 cDNA can be obtained by, for example, a so-called PCR method in which a polymerase chain reaction (hereinafter referred to as "PCR") (see Saiki, R. K., et al., Science, (1988) 239, pp. 487-489).

As in vitro synthesis of polypeptides, for example, Rapid Translation System (RTS) produced by Roche Diagnostics, Inc. can be exemplified, but not limited thereto.

Examples of prokaryotic hosts include Escherichia coli and Bacillus subtilis . To transform a host cell with a target gene, a plasmid vector containing a replicon (ie, an origin of replication from a species compatible with the host) and regulatory sequences is used to transform the host cell. In addition, the vector preferably has sequences that confer phenotype selectivity on transformed cells.

Examples of eukaryotic host cells include vertebrate cells, insect cells, and yeast cells. As vertebrate cells are often used e.g. dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin , L. A., Proc. Natl. Acad.Sci. USA (1980) 77, pp. 4126-4220), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells; ATCC: CCL- 61) etc., but they are not limited thereto.

The transformant thus obtained can be cultured according to a conventional method, and the target polypeptide can be produced intracellularly or extracellularly by culturing the transformant.

Depending on the host cells used, an appropriate medium for culture can be selected from various commonly used media. If Escherichia coli is used, for example, an LB medium to which antibiotics such as ampicillin or IPMG are added as needed can be used.

The recombinant protein produced intracellularly or extracellularly by the transformant by such culture can be isolated and purified by any of various known isolation methods, utilizing the physical or chemical properties of the protein.

Specific examples of the method include: treatment with a conventional protein precipitant, ultrafiltration, various types of liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, Dialysis, and combinations thereof.

In addition, by attaching a 6-histidine residue tag to the recombinant protein to be expressed, the protein can be efficiently purified using nickel affinity columns. Alternatively, protein A columns can be used to efficiently purify proteins by linking an IgG Fc region to the recombinant protein to be expressed.

By combining the above methods, a large amount of target polypeptide can be easily produced with high yield and high purity.

(2) Preparation of anti-Siglec-15 monoclonal antibody

Examples of antibodies that specifically bind to Siglec-15 include monoclonal antibodies that specifically bind to Siglec-15, and methods for obtaining such antibodies are described below.

The preparation of monoclonal antibodies generally requires the following steps:

(a) purification of the biopolymer to be used as an antigen;

(b) Antibody-producing cells are prepared by immunizing animals by injecting antigen, collecting blood, and measuring the antibody titer thereof to determine the time of splenectomy;

(c) preparing myeloma cells (hereinafter referred to as "myeloma");

(d) fusing the antibody-producing cells with the myeloma;

(e) screening a population of hybridomas producing the target antibody;

(f) Separation of hybridomas into single cell clones (cloning);

(g) optionally, culturing hybridomas, or animals implanted with hybridomas, to produce large quantities of monoclonal antibodies;

(h) Detecting the biological activity and binding specificity of the monoclonal antibodies thus prepared, or measuring their properties as labeling reagents, etc.

Hereinafter, the production method of the monoclonal antibody is described in detail following the above steps, but the method is not limited thereto, for example, antibody-producing cells other than spleen cells and myeloma can be used.

(a) Antigen purification

Siglec-15 or a partial peptide thereof prepared as described above can be used as an antigen.

In addition, membrane components prepared by recombinant cells expressing Siglec-15, or recombinant cells expressing Siglec-15 themselves, and partial peptides of the protein of the present invention chemically synthesized by methods known to those skilled in the art can also be used as antigen.

(b) Preparation of antibody-producing cells

The antigen obtained in step (a) is mixed with an adjuvant (such as Freund's complete or incomplete adjuvant, or potassium aluminum sulfate), and the obtained mixture is used as an immunogen to immunize experimental animals. Any animals used in known hybridoma production methods can be used as experimental animals without difficulty. Specifically, for example, mice, rats, goats, sheep, cows, horses and the like can be used. However, it is preferable to use mice or rats as animals to be immunized from the viewpoint of ease of obtaining myeloma cells fused with the extracted antibody-producing cells.

In addition, the strains of mice and rats to be used are not particularly limited, and in the case of mice, such as A, AKR, BALB/c, BDP, BA, CE, C3H, 57BL, C57BL, C57L, DBA, Different strains such as FL, HTH, HT1, LP, NZB, NZW, RF, RIII, SJL, SWR, WB and 129, in the case of rats such as Wistar, Low, Lewis, Sprague, Dawley, ACI, BN, Fischer et al.

These mice and rats are available from laboratory animal breeders/distributors, eg, CLEA Japan, Inc. and Charles River Laboratories Japan, Inc.

Among them, particularly preferred as the animal to be immunized in consideration of fusion compatibility with the following myeloma cells are: in the case of mice, the BALB/c strain; in the case of mice, Wistar and Low strains.

In addition, considering the antigenic homology between humans and mice, it is also preferable to use mice with reduced biological function (to remove autoantibodies), that is, mice with autoimmune diseases.

The age of the mice or rats at the time of immunization is preferably 5-12 weeks old, more preferably 6-8 weeks old.

To immunize animals with Siglec-15 or its recombinants, known methods such as those detailed in the following documents can be used: For example, Weir, D. M., Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher Springfield, Illinois (1964) et al.

Among these immunization methods, preferred specific methods of the present invention are as follows, for example.

That is, first, a membrane protein fraction as an antigen or a cell caused to express an antigen is intradermally or intraperitoneally administered to an animal.

However, in order to increase the immunization efficiency, the combination of the two administration routes is preferable. If the intradermal administration is performed in the first half, the immunization efficiency can be particularly improved by performing intraperitoneal administration in the second half or only at the last administration.

The administration schedule of the antigen varies depending on the kind of animal to be immunized, individual differences, and the like. In general, however, a preferred schedule of administration is one in which the antigen is administered at a frequency of 3-6 times with dosing intervals of 2-6 weeks; a more preferred schedule of administration is one in which the antigen is administered at a frequency of 3-4 times, and the dosing interval is 2-4 weeks.

In addition, the dose of antigen varies depending on the kind of animal to be immunized, individual differences, and the like. However, the dose is usually set at 0.05-5 mg, preferably about 0.1-0.5 mg.

Booster immunizations are performed 1-6 weeks, preferably 2-4 weeks, more preferably 2-3 weeks after administration of the antigen as described above.

The dose of the antigen at the time of booster immunization varies depending on the kind or size of the animal or the like. However, in the case of mice, the dose is usually set at 0.05-5 mg, preferably 0.1-0.5 mg, more preferably about 0.1-0.2 mg.

1-10 days, preferably 2-5 days, more preferably 2-3 days after the booster immunization, spleen cells or lymphocytes containing antibody-producing cells are aseptically harvested from the immunized animal.

At this point, the antibody titer is measured, and subsequent operations can be performed more efficiently if the animal with a sufficiently elevated antibody titer is used as a source of antibody-producing cells.

Examples of methods for measuring antibody titers employed here include RIA method and ELISA method, but the methods are not limited thereto.

For example, if the ELISA method is used, the antibody titer in the present invention can be measured according to the method described below.

First, a purified or partially purified antigen is adsorbed to the surface of a solid phase (such as a 96-well plate for ELISA), and then an antigen-independent protein (such as bovine serum albumin (hereinafter referred to as "BSA")) Cover the surface of the solid phase where antigen is not adsorbed. After washing the surface, the surface is exposed to a serially diluted sample (eg, mouse serum) as the primary antibody, allowing the antibodies in the sample to bind the antigen.

Furthermore, an enzyme-labeled antibody against the mouse antibody was added as a secondary antibody to bind the mouse antibody. After washing, the substrate of the enzyme is added, the change in absorbance (which occurs due to color development induced by degradation of the substrate, etc.) is measured, and based on the measurement result, the antibody titer is calculated.

According to known methods (for example, Kohler et al., Nature (1975), 256, p. 495; Kohler et al., Eur. J. Immunol. (1977), 6, p. 511; Milstein et al., Nature (1977 ), 266, p. 550; Walsh, Nature (1977), 266, p. 495), antibody-producing cells can be isolated from splenocytes or lymphocytes.

For example, in the case of splenocytes, conventional methods can be used in which antibody production is isolated by homogenizing the spleen, filtering through a stainless steel mesh to obtain the cells, and then suspending the cells in Eagle's Minimal Essential Medium (MEM). cell.

(c) Preparation of myeloma cells (hereinafter referred to as "myeloma")

The myeloma cells used for cell fusion are not particularly limited, and suitable cells can be selected from known cell lines. However, in consideration of convenience when selecting hybridomas from fused cells, it is preferable to use HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-deficient strains whose selection methods have been established.

More specifically, examples of HGPRT-deficient strains include: mouse-derived X63-Ag8 (X63), NS1-ANS/1 (NS1), P3X63-Ag8.U1 (P3U1), X63-Ag8.653 (X63. 653), SP2/0-Ag14 (SP2/0), MPC11-45.6TG1.7 (45.6TG), F0, S149/5XXO and BU.1; 210.RSY3.Ag.1.2.3 ( Y3); and human-derived U266AR (SKO-007), GM1500·GTG-A12 (GM1500), UC729-6, LICR-LOW-HMy2 (HMy2) and 8226AR/NIP4-1 (NP41).

These HGPRT-deficient strains are available from, for example, the American Type Culture Collection (ATCC) and the like.

In such as 8-azaguanine medium [by adding 8-azaguanine to RPMI supplemented with glutamine, 2-mercaptoethanol, gentamycin and fetal calf serum (hereinafter referred to as "FCS") -1640 medium], Iscove's modified Dulbecco's medium (hereinafter referred to as "IMDM"), or Dulbecco's modified Eagle's medium (hereinafter referred to as "DMEM"), etc. These cell lines were subcultured in culture medium. In this case, 3 to 4 days before performing cell fusion, cells are subcultured in a normal medium [such as ASF104 medium (manufactured by Ajinomoto Co., Ltd.) containing 10% FCS] to obtain Not less than 2×10 ⁷ cells.

(d) cell fusion

According to known methods (Weir, D. M. Handbook of Experimental Immunology Vol. I. II. III., Blackwell Scientific Publications, Oxford (1987), Kabat, E. A. and Mayer, M. M., Experimental Immunochemistry, Charles C Thomas Publisher, Springfield, Illinois (1964), etc.), the fusion between antibody-producing cells and myeloma cells can be appropriately performed without excessively reducing the viability of the cells.

As such a method, a chemical method of mixing antibody-producing cells and myeloma cells in a solution containing a polymer such as polyethylene glycol at a high concentration; a physical method using electrical stimulation, and the like can be used.

Among these methods, specific examples of chemical methods are described below.

That is, in the case where polyethylene glycol is used in a solution containing a high-concentration polymer, in a polyethylene glycol solution having a molecular weight of 1500-6000, more preferably 2000-4000, at 30-40°C, preferably 35 At a temperature of -38°C, the antibody-producing cells and the myeloma cells are mixed for 1-10 minutes, preferably 5-8 minutes.

(e) Selection of hybridoma populations

The method of selecting hybridomas obtained by the above-mentioned cell fusion is not particularly limited. Usually, the HAT (hypoxanthine, aminopterin, thymidine) selection method is used (Kohler et al., Nature (1975), 256, p. 495; Milstein et al., Nature (1977), 266, p. 550).

This method is effective when hybridomas are obtained using myeloma cells of an HGPRT-deficient line that cannot survive in the presence of aminopterin.

That is, by culturing unfused cells and hybridomas in HAT medium, only hybridomas resistant to aminopterin are selectively allowed to survive and proliferate.

(f) Separation into single cell clones (cloning)

As a method for cloning hybridomas, known methods such as the methylcellulose method, the soft agarose method, or the limiting dilution method, etc. (see, for example, Barbara, B. M. and Stanley, M. S.: Selected Methods in Cellular Immunology, W. H. Freeman and Company, San Francisco (1980)). Among these methods, the limiting dilution method is particularly preferred.

In this method, fibroblast lines from fetal rats, or feeder cells such as normal mouse splenocytes, thymocytes, or ascites cells, are seeded on microplates.

Meanwhile, the hybridomas were diluted in culture medium to obtain a cell density of 0.2-0.5 cells/0.2 ml. Add a 0.1 ml aliquot of the diluted hybridoma suspension to each well and continue culturing for approximately 2 weeks while at predetermined intervals (e.g., every 3 days) replacing approximately 1/3 of the culture medium with fresh culture medium whereby hybridoma clones can be propagated.

Hybridomas in wells for which antibody titers have been determined are subjected to cloning, for example by limiting dilution, in 2-4 repetitions. Hybridomas confirmed to have stable antibody titers were selected as hybridoma lines producing anti-Siglec-15 monoclonal antibody.

Examples of such cloned hybridoma lines include hybridoma #32A1 described in WO 09/48072. On August 28, 2008, the hybridoma #32A1 was deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology, located at: Central 6, 1-1 -1 Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), and has obtained accession number FERM BP under the name of anti-Siglec-15 Hybridoma #32A1 (anti--Siglec-15 Hybridoma #32A1) -10999. In addition, in this specification, the antibody produced by hybridoma #32A1 is referred to as "#32A1 antibody", or simply "#32A1". In addition, antibodies other than the #32A1 antibody obtained in Examples of the present specification are also indicated by antibody names in the same manner. A partial fragment containing the heavy chain variable region of antibody #32A1 has an amino acid sequence comprising amino acid residues 20-167 of SEQ ID NO: 28 in the Sequence Listing. In addition, a partial fragment containing the light chain variable region sequence of #32A1 antibody has an amino acid sequence comprising amino acid residues 21-139 of SEQ ID NO: 30 in the Sequence Listing.

(g) Production of monoclonal antibodies by culturing hybridomas

Monoclonal antibodies can be efficiently obtained by culturing hybridomas thus selected. However, hybridomas producing the monoclonal antibody of interest are preferably screened prior to culturing.

In such screening, known methods can be employed.

The antibody titer of the present invention can be measured by, for example, the ELISA method explained in the above item (b).

The hybridoma obtained by the above method can be stored in a frozen state in liquid nitrogen or in a freezer at -80°C or below.

After completion of cloning, the medium was changed from HT medium to normal medium, and hybridomas were cultured.

Large-scale cultures are performed by rotating culture using large culture flasks or by spinner culture.

Monoclonal antibodies that specifically bind to the protein of the present invention can be obtained from supernatants obtained from large-scale cultures by purification using methods known to those skilled in the art, such as gel filtration.

In addition, hybridomas containing a large amount of the monoclonal antibody of the present invention can be obtained by intraperitoneally injecting hybridomas into mice of the same strain as the hybridomas (for example, the above-mentioned BALB/c) or Nu/Nu mice to proliferate the hybridomas. ascites.

In the case of intraperitoneal administration of hybridomas, a larger amount of ascites can be obtained if mineral oil such as 2,6,10,14-tetramethylpentadecane (pristane) is administered 3 to 7 days in advance.

For example, mice of the same strain as the hybridoma are injected intraperitoneally with an immunosuppressant in advance to inactivate T cells. After 20 days, 10 ⁶ -10 ⁷ hybridoma clones were suspended in a serum-free medium (0.5 ml), and the suspension was injected intraperitoneally into mice. Generally, ascites is collected from mice when the abdomen is distended and filled with ascites.

By this method, a concentration of monoclonal antibody about 100 times higher than that in the culture solution can be obtained.

The monoclonal antibody obtained by the above method can be purified by the method described in, for example, Weir, D. M.: Handbook of Experimental Immunology Vol. I, II, III, Blackwell Scientific Publications, Oxford (1978).

That is, examples of the method include ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and affinity chromatography.

As a simple purification method, a commercially available monoclonal antibody purification kit (for example, MAbTrap GII kit manufactured by Pharmacia, Inc.) or the like can also be used.

The monoclonal antibody thus obtained has high antigen specificity for Siglec-15.

(h) Determination of monoclonal antibodies

The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows.

First, examples of identification methods include Ouchterlony method, ELISA method, and RIA method.

The Ottolony method is simple, but when the monoclonal antibody concentration is low, a concentration operation is required.

On the other hand, when using the ELISA method or the RIA method, by directly reacting the culture supernatant with the solid phase on which the antigen is adsorbed, and using antibodies corresponding to various immunoglobulin isotypes and subclasses as the secondary antibody , can identify isotypes and subclasses of monoclonal antibodies.

In addition, as a more convenient method, a commercially available identification kit (for example, a mouse typing kit produced by Bio-Rad Laboratories, Inc.) or the like can also be used.

In addition, quantitative determination of protein can be performed by the Folin Lowry method, and a calculation method based on the absorbance at 280nm [1.4 (OD280) = 1 mg immunoglobulin/ml].

(3) Other antibodies

The antibodies of the present invention include not only the above-mentioned anti-Siglec-15 monoclonal antibodies, but also artificially modified recombinant antibodies for the purpose of reducing heteroantigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. These antibodies can be prepared using known methods.

As chimeric antibodies, there can be exemplified antibodies in which antibody variable regions and constant regions are derived from different species, for example, in which a mouse- or rat-derived antibody variable region is combined with a human-derived constant region Linked chimeric antibodies (see Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984)). As an example of a chimeric antibody derived from rat anti-mouse antibody #32A1, an antibody comprising a heavy chain and a light chain having SEQ ID NO: 41 in the sequence listing can be exemplified and the light chain has an amino acid sequence comprising amino acid residues 21-237 of SEQ ID NO: 43 in the sequence listing.

As humanized antibodies, antibodies obtained by incorporating only complementarity determining regions (CDRs) into human-derived antibodies can be exemplified (see Nature (1986) 321, pp. 522-525); Grafting method, an antibody obtained by grafting a part of amino acid residues of the framework and CDR sequences into a human antibody (WO 90/07861). As an example of the humanized antibody of the rat antibody #32A1, any combination of a heavy chain containing the following heavy chain variable region and a light chain containing the following light chain variable region can be exemplified. The variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 51, amino acid residues 20-140 of SEQ ID NO: 53, amino acid residue 20 of SEQ ID NO: 55 in the Sequence Listing -140, amino acid residues 20-140 of SEQ ID NO: 57, amino acid residues 20-140 of SEQ ID NO: 59, amino acid residues 20-140 of SEQ ID NO: 99, amino acid residues of SEQ ID NO: 101 Bases 20-140, or amino acid residues 1-121 of any one of the amino acid sequences represented by SEQ ID NO: 72-77, said light chain variable region comprising an amino acid sequence comprising SEQ ID NO in the Sequence Listing : amino acid residues 21-133 of 61, amino acid residues 21-133 of SEQ ID NO: 63, amino acid residues 21-133 of SEQ ID NO: 65, amino acid residues 21-132 of SEQ ID NO: 67, SEQ ID NO: 67 Amino acid residues 21-133 of ID NO: 69, amino acid residues 21-133 of SEQ ID NO: 71, amino acid residues 21-133 of SEQ ID NO: 103, amino acid residues 21-133 of SEQ ID NO: 105, Or amino acid residues 1-113 of any one of the amino acid sequences represented by SEQ ID NO: 78-96.

As a preferred combination, an antibody comprising a heavy chain comprising the following heavy chain variable region and a light chain comprising the following light chain variable region comprising SEQ ID NO: : 51, and the light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 61, or the heavy chain variable region comprises an amino acid sequence comprising SEQ ID NO: 61 An amino acid sequence of amino acid residues 20-140 of ID NO: 53, and said light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 63, or said heavy chain variable region comprises An amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 55, and said light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 65, or said heavy chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 55, and the light chain variable region comprises an amino acid sequence comprising amino acid residues 21-132 of SEQ ID NO: 67, or the heavy chain may The variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 57, and the light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 69, or the heavy The chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 59, and the light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 71, or the The heavy chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 99, and the light chain variable region comprises an amino acid sequence comprising amino acid residues 21-133 of SEQ ID NO: 69, Or the heavy chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 101 and the light chain variable region comprises amino acids comprising amino acid residues 21-133 of SEQ ID NO: 69 sequence, or the heavy chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 99, and the light chain variable region comprises amino acid residues 21-133 comprising SEQ ID NO: 103 or the heavy chain variable region comprises an amino acid sequence comprising amino acid residues 20-140 of SEQ ID NO: 99, and the light chain variable region comprises amino acid residues 21-140 comprising SEQ ID NO: 105 133 amino acid sequence.

As a more preferable combination, an antibody comprising a heavy chain having an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 51 and a light chain comprising SEQ ID NO: 51 can be exemplified. : an amino acid sequence of amino acid residues 21-238 of 61, or the heavy chain has an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 53, and the light chain has an amino acid sequence comprising SEQ ID NO: 63 An amino acid sequence of residues 21-238, or the heavy chain has an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 55, and the light chain has an amino acid sequence comprising amino acid residues 21-470 of SEQ ID NO: 65 238, or the heavy chain has an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 55, and the light chain has an amino acid sequence comprising amino acid residues 21-237 of SEQ ID NO: 67, Or the heavy chain has an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 57, and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 69, or the heavy chain chain has an amino acid sequence comprising amino acid residues 20-470 of SEQ ID NO: 59, and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 71, or the heavy chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 71, or the heavy chain has an amino acid sequence comprising ID NO:99 amino acid residues 20-466, and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO:69, or the heavy chain has an amino acid sequence comprising SEQ ID NO:101 and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 69, or the heavy chain has an amino acid residue comprising SEQ ID NO: 99 20-466, and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 103, or the heavy chain has an amino acid sequence comprising amino acid residues 20-466 of SEQ ID NO: 99 an amino acid sequence, and the light chain has an amino acid sequence comprising amino acid residues 21-238 of SEQ ID NO: 105.

However, the humanized antibody derived from the #32A1 antibody is not limited to the above-mentioned humanized antibody as long as the humanized antibody has all the 6 types of CDR sequences of #32A1 and has the activity of inhibiting osteoclast formation. In addition, the heavy chain variable region of the #32A1 antibody has: CDRH1 (DYFMN) comprising the amino acid sequence represented by SEQ ID NO: 44, CDRH2 (QIRNKIYTYATFYAESLEG) comprising the amino acid sequence represented by SEQ ID NO: 45, and Any one of the CDRH2 (QIRNKIYTYATFYA) represented by NO: 97, and the CDRH3 (SLTGGDYFDY) comprising the amino acid sequence represented by SEQ ID NO: 46. CDRH2 represented by SEQ ID NO: 45 is defined according to Kabat (SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST VOL. I, 5th edition (1991)). The CDRH2 represented by SEQ ID NO: 97 is shortened by 5 residues at the C-terminus compared to the Kabat definition. In the heavy chain sequence containing this CDRH2, the rat-derived CDR sequence is made shorter and incorporates more human framework sequences, therefore, when it is administered to humans, the possibility of being recognized as a heterologous antigen lower. In addition, the light chain variable region of the #32A1 antibody has: CDRL1 (RASQSVTISGYSFIH) comprising the amino acid sequence represented by SEQ ID NO: 47, CDRL2 (RASNLAS) comprising the amino acid sequence represented by SEQ ID NO: 48, and CDRL2 (RASNLAS) comprising the amino acid sequence represented by SEQ ID NO: 48 CDRL3 (QQSRKSPWT) of the amino acid sequence represented by ID NO: 49.

Furthermore, antibodies of the invention include human antibodies. Human anti-Siglec-15 antibody refers to a human antibody having only antibody gene sequences derived from human chromosomes. Human anti-Siglec-15 antibodies can be obtained by a method using a human antibody-producing mouse having a human chromosomal fragment containing the heavy and light chain genes of the human antibody (see Tomizuka, K. et al., Nature Genetics (1997) 16, pp. 133-143; Kuroiwa, Y. et al., Nucl. AcidsRes. (1998) 26, pp. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, pp. 69-73 (eds. Kitagawa, Y., Matuda, T. and Iijima, S.), Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc.Natl. Acad. Sci. USA (2000) 97, pp. 722-727, etc.).

Specifically, such human antibody-producing mice can be established as follows. Genetically modified animals in which the endogenous immunoglobulin heavy and light chain gene loci have been disrupted and, alternatively, transformed via yeast artificial chromosome (YAC) are created by making knockout and transgenic animals, and mating these animals. ) vectors etc. have introduced human immunoglobulin heavy chain and light chain gene loci.

In addition, according to genetic engineering techniques, by using cDNAs respectively encoding the heavy chain and light chain of such human antibodies, and preferably using vectors containing the cDNAs, transforming eukaryotic cells, and culturing transformants producing recombinant human monoclonal antibodies, by This antibody can also be obtained from the culture supernatant.

Here, for example, eukaryotic cells, preferably mammalian cells, such as CHO cells, lymphocytes or myeloma cells can be used as hosts.

In addition, methods for obtaining phage display-derived human antibodies screened from human antibody libraries are also known (see Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science. (2002) 43 (7), pp. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), pp. 189-203; Siriwardena, D. et al., Opthalmology (2002) 109 (3), pp. 427-431, et al. ).

For example, a phage display method can be used in which variable regions of human antibodies are expressed on the surface of phages as single-chain antibodies (scFv), and phages that bind antigens are selected (Nature Biotechnology (2005), 23, (9), pp. 1105- 1116 pages).

By analyzing the genes of phage selected based on binding to the antigen, the DNA sequence encoding the variable region of the human antibody that binds the antigen can be determined.

If the DNA sequence of an antigen-binding scFv is determined, a human antibody can be obtained by preparing an expression vector having the sequence and introducing the vector into an appropriate host to express it (WO 92/01047, WO 92/20791, WO 93 /06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994) 12, pp. 433-455, Nature Biotechnology (2005) 23 (9), p. pp. 1105-1116).

By the method described in Example 25 and the like, the antigen-binding property of the antibody obtained by the above method can be evaluated, and a preferable antibody can be selected. As an example of another index when comparing the properties of antibodies, the stability of antibodies can be mentioned. Differential Scanning Calorimetry (DSC), shown in Example 33, is an instrument capable of rapidly and accurately measuring the thermal denaturation midpoint temperature (Tm), a favorable indicator of the relative structural stability of proteins . Differences in thermal stability can be compared by measuring the Tm value using DSC and comparing this value. It is known that the storage stability of antibodies shows some correlation with the thermal stability of antibodies (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, pp. 265-273), using thermal stability as an index, Preferred antibodies can be selected. Examples of other criteria for selecting antibodies include the following: higher production in appropriate host cells and lower aggregation in aqueous solutions. For example, an antibody showing the highest yield does not always show high thermal stability, and therefore, it is necessary to select an antibody most suitable for administration to humans by making a comprehensive evaluation based on the above-mentioned indicators.

In addition, methods are also known in which the full-length heavy and light chain sequences of antibodies are joined using appropriate linkers, thereby obtaining single-chain immunoglobulins (Lee, H-S, et al., Molecular Immunology (1999) 36, pp. 61-71; Shirrmann, T. et al., mAbs (2010), 2, (1) pp. 1-4). By dimerizing such single-chain immunoglobulins, the resulting dimer can have a structure and activity similar to that of an antibody that is itself a tetramer. Furthermore, an antibody of the invention may be an antibody having a single heavy chain variable region and no light chain sequence. Such antibodies are called single-domain antibodies (sdAbs) or nanobodies, and indeed, they have been observed in llamas and alpacas and have been reported to have antigen-binding affinity (Muyldemans S. et al., Protein Eng. (1994) 7(9), 1129-35, Hamers-Casterman C. et al., Nature (1993) 363 (6428) 446-8). It can be understood that the above-mentioned antibodies are types of functional fragments of antibodies according to the present invention.

Furthermore, by controlling glycosylation, in which glycans are bound to the antibodies of the invention, it is possible to enhance antibody-dependent cellular cytotoxic activity. Regarding techniques for controlling glycosylation of antibodies, WO 99/54342, WO 00/61739, WO 02/31140 and the like are known. However, the technique is not limited thereto.

In the case of first isolating an antibody gene and then introducing the gene into an appropriate host to produce an antibody, a combination of an appropriate host and an appropriate expression vector can be used. Specific examples of antibody genes include a combination of a gene encoding the heavy chain sequence of the antibody described in this specification and a gene encoding the light chain sequence thereof. When transforming a host cell, it is possible to insert the heavy chain sequence gene and the light chain sequence gene into the same expression vector, and it is also possible to insert them into different expression vectors, respectively. In the case of using eukaryotic cells as hosts, animal cells, plant cells and eukaryotic microorganisms can be used. As animal cells, there can be exemplified (1) mammalian cells, for example, dihydrofolate reductase of ape COS cells (Gluzman, Y., Cell, (1981) 23, pp. 175-182, ATCC CRL-1650)- Defective strain (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), mouse fibroblast NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells; ATCC: CCL-61). Furthermore, in the case of using prokaryotic cells, for example, Escherichia coli and Bacillus subtilis can be exemplified. The antibody can be obtained by transforming, introducing the target antibody gene into these cells, and culturing the thus transformed cells in vitro. In the above-mentioned culture method, the yield may sometimes vary depending on the sequence of the antibody. Therefore, by using the yield as an index, it is possible to select an antibody that can be easily produced as a drug among antibodies having comparable binding activity.

The isotype of the antibody of the present invention is not limited, and examples thereof include IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgD, and IgE, and preferred examples thereof include IgG and IgM, and further, More preferable examples thereof include IgG1 and IgG2.

In addition, the antibody of the present invention may be a functional fragment of an antibody having an antigen-binding site of the antibody or a modified fragment thereof. Fragments of antibodies can be obtained by treating antibodies with proteases such as papain or pepsin, or modifying antibody genes according to genetic engineering techniques, and expressing the modified genes in appropriate cultured cells. Among these antibody fragments, a fragment having all or part of the functions of a full-length antibody molecule can be referred to as a functional fragment of the antibody. The functions of the antibody generally include antigen-binding activity, activity to neutralize antigen activity, activity to enhance antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, and complement-dependent cellular cytotoxicity. cytotoxic activity. The function of the antibody functional fragment according to the present invention is the binding activity to Siglec-15, preferably the activity of inhibiting osteoclast formation, more preferably the activity of inhibiting the cell fusion process of osteoclasts.

Examples of antibody fragments include Fab, F(ab')2, Fv, single-chain Fv (scFv, in which the Fv molecules of the heavy and light chains are linked by an appropriate linker), diabodies, linear antibodies, and antibody fragments consisting of multispecific antibodies. In addition, Fab' (a monovalent fragment in the variable region of an antibody obtained by treating F(ab')2 under reducing conditions) is also considered an antibody fragment.

Furthermore, antibodies of the invention may be multispecific antibodies specific for at least two different antigens. Typically, such molecules bind two antigens (i.e., bispecific antibodies), however, "multispecific antibody" as used herein includes antibodies with specificity for two or more (e.g., 3) antigens .

Multispecific antibodies of the invention can be full-length antibodies or fragments of such antibodies (e.g., F(ab')2 bispecific antibodies). Bispecific antibodies can be produced by linking the heavy and light chains (HL pairs) of two types of antibodies, or by fusing hybridomas producing different monoclonal antibodies to produce fusion cells that produce bispecific antibodies, or bispecific antibodies can be produced Specific antibodies (Millstein et al., Nature (1983) 305, pp. 537-539).

Antibodies of the invention may be single chain antibodies (also known as scFv). Single-chain antibodies can be obtained by linking the heavy and light chain variable regions of the antibody via a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113 (Eds. Rosenburg and Moore, Springer Verlag, New York, pp. 269-315) (1994), Nature Biotechnology (2005), 23, pp. 1126-1136). In addition, BiscFv fragments generated by linking two scFv molecules via a polypeptide linker can also be used as bispecific antibodies.

Methods of making single chain antibodies are known in the art (see, eg, US Patent Nos. 4,946,778, 5,260,203, 5,091,513, 5,455,030, etc.). In this scFv, the variable region of the heavy chain and the variable region of the light chain are linked by a linker which does not form a conjugate, preferably by a polypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) , 85, pp. 5879-5883). In scFv, the heavy and light chain variable regions can be derived from the same antibody or from different antibodies.

As a polypeptide linker for linking variable regions, for example, a given single-chain peptide consisting of 12 to 19 residues is used.

DNA encoding scFv can be obtained by using as a template DNA encoding the entire amino acid sequence or a desired partial amino acid sequence (selected from the heavy chain or heavy chain variable region and the light chain or light chain variable region of the above-mentioned antibodies), And using the primer pair that defines its two ends, amplify by PCR method, and by combining the DNA encoding the polypeptide joint part and the primer pair that defines its two ends, so as to connect the two ends of the heavy chain and the light chain respectively, and further amplify .

In addition, after preparing DNA encoding scFv, an expression vector containing the DNA and a host transformed with the expression vector can be obtained according to conventional methods. Also, by using the obtained host, scFv can be obtained according to a conventional method. By obtaining a gene and expressing the gene in the same manner as above, antibody fragments thereof can be produced in a host.

Antibodies of the invention can be multimerized to increase affinity for antigen. Antibodies to be multimerized may be one class of antibodies, or multiple antibodies recognizing multiple epitopes of the same antigen. Examples of methods for multimerizing antibodies include binding IgG CH3 domains to two scFv molecules, binding streptavidin, introducing a helix-turn-helix motif, and the like.

The antibody of the present invention may be a polyclonal antibody, which is a mixture of multiple types of anti-Siglec-15 antibodies with different amino acid sequences. As an example of a polyclonal antibody, a mixture of multiple types of antibodies having different CDRs can be exemplified. Cultivating a mixture of cells producing different antibodies, and purifying antibodies from the resulting culture can be used as such polyclonal antibodies (see WO 2004/061104).

Antibodies conjugated to any of various types of molecules such as polyethylene glycol (PEG) can also be used as modified antibodies.

Furthermore, the antibody of the present invention may be in the form of a conjugate (immunoconjugate) formed between any such antibody and another drug. Examples of such antibodies include those in which the antibody is conjugated to a radioactive substance or a compound having a pharmacological action (Nature Biotechnology (2005) 23, pp. 1137-1146).

The resulting antibody can be purified to homogeneity. Antibody isolation and purification can be performed using conventional protein isolation and purification methods. For example, antibodies can be isolated and purified by appropriate selection and combination of column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Strategies for protein Purification and Charcterization: A Laboratorytoy Course Manual, edited by Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press (1996); Antibodies: A Laboratory Manual. Edited by Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), but the method is not limited thereto.

Examples of such chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.

These chromatography methods can be performed using liquid chromatography, such as HPLC or FPLC.

As the column used for the affinity chromatography, a protein A column and a protein G column may be exemplified.

For example, as a column using a protein A column, Hyper D, POROS, Sepharose FF (Pharmacia), etc. can be mentioned.

In addition, antibodies can also be purified by using a carrier on which an antigen is immobilized, utilizing the binding property of the antibody to the antigen.

4. Drugs containing anti-Siglec-15 antibodies

From the anti-Siglec-15 antibody obtained by the method described in the above item "3. Preparation of anti-Siglec-15 antibody", an antibody that neutralizes the biological activity of Siglec-15 can be obtained. These antibodies that neutralize the biological activity of Siglec-15 can inhibit the biological activity of Siglec-15 in vivo, that is, the differentiation and/or maturation of osteoclasts, and thus can be used as drugs for the abnormal differentiation and/or maturation of osteoclasts. A therapeutic agent and/or a preventive agent for abnormal bone metabolism caused by/or maturation. Abnormal bone metabolism can be any disorder characterized by net bone loss (osteopenia or osteolysis). In general, treatment and/or prophylaxis with anti-Siglec-15 antibodies should be used in situations where inhibition of bone resorption is desired. Examples of abnormal bone metabolism that can be treated and/or prevented with an anti-Siglec-15 antibody include: osteoporosis (postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis, or osteoporosis with rheumatoid arthritis), bone destruction with rheumatoid arthritis, cancerous hypercalcemia, bone disease with multiple myeloma or cancer Metastatic bone destruction, giant cell tumor, osteopenia, tooth loss due to periodontitis, periarticular osteolysis, bone destruction in chronic osteomyelitis, Paget's disease of bone, renal osteodystrophy, and osteogenesis Insufficient, however, abnormal bone metabolism is not limited thereto as long as it is a disease with net bone loss by osteoclasts. Examples of the anti-Siglec-15 antibody used as the above drug include chimeric antibody and humanized antibody prepared from #32A1 antibody by the method described in the item of 3. "(3) Other antibodies". In addition, chimeric antibodies, humanized antibodies, and human antibodies having the same epitope as #32A1 antibody can also be used as medicines. Whether a certain anti-Siglec-15 antibody has the same epitope as the #32A1 antibody can be confirmed by observing whether these antibodies bind to the same specific partial peptide of Siglec-15. In addition, if a certain anti-Siglec-15 antibody competes with #32A1 antibody for binding to Siglec-15, it can also be judged that these antibodies have the same epitope.

The in vitro activity of anti-Siglec-15 antibodies to neutralize the biological activity of Siglec-15 can be determined by, for example, inhibiting the activity of cells overexpressing Siglec-15 to differentiate into osteoclasts. For example, the addition of anti-Siglec-15 antibody to RAW264.7 cells or RAW 264 cells (mouse monocyte-derived cell line) at different concentrations can measure the inhibition of RANKL or TNF-α stimulation to osteoclasts. differentiation activity. In addition, the addition of anti-Siglec-15 antibody to bone marrow-derived primary cultured cells at different concentrations can determine the activity of stimulation of RANKL, TNF-α, or activated vitamin _D3 to inhibit osteoclast differentiation. In addition, anti-Siglec-15 was added to normal human osteoclast precursor cells (Normal Human Natural Osteoclast Precursor Cells, available from Sanko Junyaku Co., Ltd., Cat. No. 2T-110) at different concentrations Antibodies that can be used to measure the activity of stimulation of RANKL or M-CSF to inhibit osteoclast differentiation. Such an inhibitory effect on osteoclast differentiation can be determined by using inhibition of tartrate-resistant acid phosphatase (TRAP) activity as an index. In addition, the inhibitory effect on osteoclast differentiation can also be determined by using inhibition of TRAP-positive multinucleated osteoclast formation (ie, inhibition of osteoclast cell fusion) as an index. Furthermore, in lacunae assay experiments using femur- and/or tibial-derived cells (Takada et al., Bone and Mineral, (1992) 17, 347-359), by injecting femur- and/or tibial - Anti-Siglec-15 antibody added to the derived cells and observation of lacuna formation on dentine sections allow determination of the in vitro activity of osteoclasts in inhibiting bone resorption. As a system for measuring the in vitro activity of inhibiting osteoclast-induced bone resorption, plates coated with europium-conjugated human collagen can also be used. The in vivo therapeutic or preventive effect of the anti-Siglec-15 antibody on abnormal bone metabolism using an experimental animal can be confirmed by administering the anti-Siglec-15 antibody to an osteoporosis animal model or a transgenic animal overexpressing Siglec-15, and Measure any changes in osteoclasts.

The antibody neutralizing the biological activity of Siglec-15 obtained in this way can be used as a medicine, especially as a pharmaceutical composition for treating or preventing abnormal bone metabolism such as osteoporosis, concomitant rheumatoid arthritis Inflammatory bone destruction, or bone destruction accompanied by cancerous bone metastasis, or as an antibody for immunological diagnosis of such diseases.

A major challenge in the treatment of rheumatoid arthritis (RA) is the bone loss that accompanies the disease. Osteoclasts have been reported to play a major role in this bone loss that accompanies RA. RANKL and TNF-α are considered to be the most important cytokines for osteoclast induction (differentiation and maturation) and activation, and are responsible for bone destruction in RA (Romas E. et al., Bone 30, pp. 340-346, 2002). OCIF/OPG, a decoy receptor for RANKL, inhibited RANKL-induced osteoclastogenesis, but not TNF-α-induced osteoclastogenesis. On the other hand, the anti-Siglec-15 antibody according to the present invention effectively inhibits osteoclastogenesis induced by RANKL and TNF-α. Therefore, it is expected that the anti-Siglec-15 antibody of the present invention can inhibit TNF-α-induced bone loss and bone destruction in RA and the like more strongly than RANKL blockers (OCIF/OPG, anti-RANKL antibody, etc.).

As an example, for the treatment or prevention of abnormal bone metabolism, an anti-Siglec-15 antibody can be administered alone or in combination with at least one other therapeutic agent for a bone-related disease. As another example, an anti-Siglec-15 antibody can be administered in combination with a therapeutically effective amount of a therapeutic agent for abnormal bone metabolism. Examples of therapeutic agents that can be administered in combination with an anti-Siglec-15 antibody include, but are not limited to: bisphosphonates (e.g., alendronate, etidronate, ibandronate, icadronate , pamar diphosphate, risedronate and zoledronate), active vitamin D ₃ , calcitonin and its derivatives, hormones such as estradiol, SERMs (selective estrogen receptor modulators) , ipriflavone, vitamin K2 ₍ menaquinone), calcium preparations, PTH (parathyroid hormone), NSAIDs (eg, celecoxib and rofecoxib), soluble TNF Receptor (eg, etanercept), anti-TNF-alpha antibody or functional fragment of said antibody (eg, infliximab), anti-PTHrP (parathyroid hormone-related protein) antibody or said antibody functional fragments of IL-1 receptor antagonists (for example, anakinra), anti-IL-6 receptor antibodies or functional fragments of said antibodies (for example, tocilizumab), anti-RANKL antibodies or Functional fragments of such antibodies (eg, denosumab) and OCIF (osteoclastogenesis inhibitory factor). Depending on the state of abnormal bone metabolism or the desired degree of treatment and/or prevention, 2 or 3 or more drug types can be administered, and these drugs can be supplied together by being encapsulated in the same preparation. These drugs and anti-Siglec-15 antibodies can also be delivered together by encapsulating them in the same formulation. Furthermore, these drugs can also be supplied together by packaging them as a kit for treatment and/or prevention. In addition, these drugs and anti-Siglec-15 antibodies can be supplied separately. In the case of administration in gene therapy, the gene of the proteinaceous bone disease therapeutic agent and the gene of the anti-Siglec-15 antibody can be inserted downstream of the same promoter region or different promoter regions, and can be introduced into the same vector or in different carriers.

By conjugating a bone disease therapeutic agent to an anti-Siglec-15 antibody or fragment thereof, it can be prepared as described in M. C. Garnet "Targeted drug conjugates: principles and progress", Advanced Drug Delivery Reviews, (2001) 53, 171-216 targeted drug conjugates. For this purpose, any antibody fragments other than antibody molecules can be used as long as they do not completely lose the ability to recognize osteoclasts, examples of which include fragments such as Fab, F(ab')2 and Fv. In the present invention, antibodies and fragments can also be used in the same manner. Conjugates of anti-Siglec-15 antibodies or fragments thereof with therapeutic agents for bone disease can be in any of a variety of forms described in: M. C. Garnet "Targeted drug conjugates: principles and progress", Advanced Drug Delivery Reviews, (2001) 53, 171-216, G. T. Hermanson “Bioconjugate Techniques” Academic Press, California (1996), Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, et al. 55-123 . That is, there can be exemplified a conjugate in which an anti-Siglec-15 antibody and a bone disease therapeutic agent are chemically directly conjugated to each other, or conjugated via a spacer such as an oligopeptide, and via an appropriate drug carrier. formed conjugates. Examples of drug carriers include liposomes and water-soluble polymers. More specific examples of a conjugate formed via such a drug carrier include: a conjugate in which an antibody and a bone disease therapeutic agent are incorporated into a liposome, and the liposome and the antibody are conjugated to each other; and such Conjugates in which bone disease therapeutic agents are chemically directly conjugated to water-soluble polymers (compounds with a molecular weight of about 1,000-100,000), or via spacers such as oligopeptides, and antibodies are combined with water-soluble polymers conjugate. Conjugation of antibodies (or fragments thereof) to bone disease therapeutic agents or drug carriers (such as liposomes or water-soluble polymers) can be achieved by methods known to those skilled in the art, such as described in Methods: G. T. Hermanson "Bioconjugate Techniques" Academic Press, California (1996), Putnam and J. Kopecek "Polymer Conjugates with Anticancer Activity" Advances in Polymer Science (1995) 122, 55-123. Incorporation of bone disease therapeutic agents into liposomes can be achieved by methods known to those skilled in the art, such as those described in D. D. Lasic "Liposomes: From Physics to Applications" Elsevier Science Publishers B. V., Amsterdam (1993) et al. Conjugation of bone disease therapeutic agents to water-soluble polymers can be accomplished by methods known to those skilled in the art, such as those described in D. Putnam and J. Kopecek "Polymer Conjugates with Anticancer Activity "Advances in Polymer Science (1995) 122, 55-123. In addition to the above methods, conjugates of antibodies (or fragments thereof) and proteinaceous bone disease therapeutic agents (or fragments thereof) can be prepared by genetic engineering by methods known to those skilled in the art.

The present invention also provides a pharmaceutical composition, which contains a therapeutically and/or prophylactically effective amount of anti-Siglec-15 antibody and pharmaceutically acceptable diluents, carriers, solubilizers, emulsifiers, preservatives and/or adjuvants.

The present invention also provides a pharmaceutical composition, which contains a therapeutically and/or preventively effective amount of an anti-Siglec-15 antibody, a therapeutically and/or preventively effective amount of at least one bone disease therapeutic agent, and a pharmaceutically acceptable diluent, Carriers, solubilizers, emulsifiers, preservatives and/or adjuvants. Examples of bone disease therapeutics include, but are not limited to: bisphosphonates (e.g., alendronate, etidronate, ibandronate, icadronate, pamadiphosphate, riser phosphonates and zoledronates), active vitamin D ₃ , calcitonin and its derivatives, hormones such as estradiol, SERMs (selective estrogen receptor modulators), ipriflavone, vitamin K ₂ ( Menadione), calcium preparations, PTH (parathyroid hormone), NSAIDs (eg, celecoxib and rofecoxib), soluble TNF receptors (eg, etanercept ), anti-TNF-α antibodies or functional fragments of said antibodies (e.g., infliximab), anti-PTHrP (parathyroid hormone-related protein) antibodies or functional fragments of said antibodies, IL-1 receptor Antagonists (eg, anakinra), anti-IL-6 receptor antibodies or functional fragments of such antibodies (eg, tocilizumab), anti-RANKL antibodies or functional fragments of such antibodies (eg, denosumab) and OCIF (osteoclastogenesis inhibitory factor).

Substances acceptable in the pharmaceutical composition according to the present invention for use in the formulation are preferably non-toxic to the person to whom the pharmaceutical composition is administered in their administered amounts and concentrations.

The pharmaceutical compositions of the present invention may contain pharmaceutical substances that alter or maintain pH, osmotic pressure, viscosity, clarity, color, isotonicity, sterility, stability, solubility, release rate, absorption rate, and their permeability. Examples of such pharmaceutical substances include, but are not limited to: amino acids such as glycine, alanine, glutamine, asparagine, arginine, and lysine; antimicrobial agents; antioxidants such as ascorbic acid, sodium sulfate, and sulfite Sodium hydrogen; buffers such as phosphate, citrate, borate buffers, sodium bicarbonate, and Tris-HCl solutions; bulking agents such as mannitol and glycine; chelating agents such as ethylenediamine tetraacetate (EDTA); complexing agents Such as caffeine, polyvinylpyrrolidine, beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin; bulking agents such as glucose, mannose and dextrin; other carbohydrates such as monosaccharides and disaccharides; coloring flavoring agents; diluents; emulsifiers; hydrophilic polymers such as polyvinylpyrrolidine; preservatives such as low molecular weight polypeptides, salt-forming counterions, benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol , methylparaben, propylparaben, chlorhexidine, sorbic acid and hydrogen peroxide; solvents such as glycerin, propylene glycol and polyethylene glycol; sugar alcohols such as mannitol and sorbitol; suspending agents; topical Active agents such as sorbitan esters, polysorbates including polysorbate 20 and polysorbate 80, triton (Triton), tromethamine, lecithin, and cholesterol; stability enhancers such as sucrose and sorbitol; elasticity enhancers such as sodium chloride, potassium chloride, and mannitol and sorbitol; transport agents; excipients; and/or pharmaceutical adjuvants. The amount of these substances added for pharmaceutical use is preferably 0.01-100 times the weight of the anti-Siglec-15 antibody, particularly preferably 0.1-10 times. Those skilled in the art can appropriately determine the preferred composition of the pharmaceutical composition in the formulation according to the disease to which the composition is applied, the route of administration to which it is applied, and the like.

Excipients and carriers in pharmaceutical compositions can be in liquid or solid form. Suitable excipients or carriers may be water for injection, physiological saline, artificial cerebrospinal fluid or other substances commonly used in parenteral administration. In addition, neutral physiological saline or serum albumin-containing physiological saline can also be used as a carrier. The pharmaceutical composition may contain Tris buffer at pH 7.0-8.5, acetate buffer at pH 4.0-5.5 or citrate buffer at pH 3.0-6.2. Additionally, such buffers can be supplemented with sorbitol or another compound. Examples of the pharmaceutical composition of the present invention include a pharmaceutical composition comprising an anti-Siglec-15 antibody, and a pharmaceutical composition comprising an anti-Siglec-15 antibody and at least one bone disease therapeutic agent. The pharmaceutical composition of the present invention is prepared as a lyophilized product or in liquid form as a drug having a selected composition and desired purity. Appropriate excipients (such as sucrose) can also be used to form the pharmaceutical composition containing anti-Siglec-15 antibody and the pharmaceutical composition containing anti-Siglec-15 antibody and at least one therapeutic agent for abnormal bone metabolism into a lyophilized product .

The pharmaceutical compositions of the present invention may be formulated for parenteral administration or for gastrointestinal absorption via oral administration. The composition and concentration of the formulation can be determined according to the method of administration. The higher the affinity of the anti-Siglec-15 antibody contained in the pharmaceutical composition of the present invention to Siglec-15, that is to say, the lower its dissociation constant (Kd value) for Siglec-15, the anti-Siglec-15 15 Antibodies can exhibit more drug potency (even at reduced doses given to humans). Therefore, based on this consideration, the dosage of the pharmaceutical composition of the present invention in humans can also be determined. Regarding the dose, in the case of administering the human anti-Siglec-15 antibody to humans, the antibody can be administered at a dose of about 0.1-100 mg/kg (once every 1-180 days).

Examples of dosage forms of the pharmaceutical composition of the present invention include injections including infusion, suppositories, nasal preparations, sublingual preparations, and percutaneous absorption preparations.

Example

Hereinafter, the present invention is described more specifically with reference to Examples, however, the present invention is not limited thereto. It should be pointed out that, unless otherwise specified, each operation related to genetic manipulation in the following examples is based on "Molecular Cloning" (Sambrook, J., Fritsch, E. F. and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989 published), or in the case of using commercially available reagents or kits, use them according to the accompanying instructions.

Example 1 Preparation of soluble mouse Siglec-15 protein expression construct

The partial nucleotide sequence of the extracellular domain encoding the mouse Siglec-15 protein is represented by SEQ ID NO: 5 in the sequence listing, and its amino acid sequence is represented by SEQ ID NO: 6 in the sequence listing. By utilizing such a partial sequence, soluble mouse Siglec-15 protein can be produced in the culture supernatant of animal cells or the like.

a) Amplification of soluble mouse Siglec-15 gene by PCR

According to a conventional method, an oligonucleotide having the sequence 5'-ggggacaagt ttgtacaaaa aagcaggcttcaccATGGAG GGGTCCCCTCC AACTC-3' (mSiglec-15-ECD-F: SEQ ID NO: 7 in the sequence listing) and an oligonucleotide having the sequence 5'-ggggaccact ttgtacaaga were synthesized. aagctgggtc TCCGGGGGCG CCGTGGAAGCGGAAC-3' (mSiglec-15-ECD-R: SEQ ID NO: 8 in the Sequence Listing) oligonucleotide as a primer for amplifying mouse Siglec-15 extracellular domain cDNA by PCR. In addition, these primers were designed as amplification primers for the production of gateway entry clones to add attB1 sequence in mSiglec-15-ECD-F and attB2 sequence in mSiglec-15-ECD-R. PCR was carried out using this primer combination and a polynucleotide containing the open reading frame sequence of mouse Siglec-15 as a template according to a conventional method. The conditions of the thermal cycler were set as follows: after heating at 94°C for 5 minutes, repeat the temperature cycle of "94°C for 0.5 minutes, 55°C for 0.5 minutes, and 68°C for 1.5 minutes" 15 times, then heat at 68°C for 5 minutes, and then Incubate at 4°C.

b) Preparation of entry clones by Gateway BP reaction

An entry clone in which mouse Siglec-15 extracellular domain cDNA was integrated by Gateway technology (Invitrogen, Inc.) using a site-specific recombination system of bacteriophage lambda was prepared by the following method. First, between the PCR product having the attB sequence prepared in a) at both ends and pDNOR221 (manufactured by Invitrogen, Inc., which is a donor vector having attP sequence), a BP reaction was performed using BP Clonase. Using this reaction solution, Escherichia coli DH10B was transformed, colony PCR was performed for the drug-resistant clone, and the size of the insert was confirmed. Then, for clones confirmed to have an insert of the correct size, sequence analysis of the total DNA sequence of the insert was performed. As a result, an incoming clone completely identical to the target nucleotide sequence (SEQ ID NO: 5 in the Sequence Listing) encoding the extracellular domain of the mouse Siglec-15 protein was obtained.

c) Preparation of expression clones by Gateway LR reaction

An expression clone in which mouse Siglec-15 extracellular domain cDNA was integrated by Gateway technology (Invitrogen, Inc.) using a λ bacteriophage site-specific recombination system was prepared by the following method. The entry clone prepared in b) contains an insert with attL sequences at both ends. Between this entry clone and the two types of destination vectors with attR sequences, an LR reaction was performed using LR Clonase. Furthermore, as the destination vector, two types of destination vectors were used: pDONM designed to add a V5 epitope tag and 6×His tag to the C-terminus of the insert, and phIgFc designed to add a human Fc tag to the C-terminus of the insert. Escherichia coli DH10B was transformed by using the reaction solution obtained by the LR reaction, colony PCR was performed on the obtained drug-resistant clone, and the size of the insert was confirmed. Then, for the clone confirmed to have an insert of the correct size, sequence analysis was performed on both ends from the insert side to the vector side.

Primer sequences for sequence analysis:

5'-tgcgtgaagg tgcagggcag-3' (mSiglec-15-ECD-seq-upstm: SEQID NO: 9 in the sequence listing)

with

5'-cctcgcctgg tcgggtc-3' (mSiglec-15-ECD-seq-dnstm: SEQ ID NO: 10 in the Sequence Listing).

As a result of the sequence analysis, expression clones (soluble mouse Siglec-15/pDONM and soluble mouse Siglec-15/phIgFc) in which correct recombination of pDONM and phIgFc occurred were respectively obtained. By transfecting soluble mouse Siglec-15/pDONM into animal cells, etc., the mRNA having the base sequence represented by SEQ ID NO: 11 in the sequence listing is transcribed, and is translated into having the SEQ ID NO in the sequence listing :12 represents the amino acid sequence of the protein (mouse Siglec-15-His). In addition, by transfecting soluble mouse Siglec-15/phIgFc into animal cells and the like, mRNA having a base sequence represented by SEQ ID NO: 13 in the Sequence Listing is transcribed and translated into a sequence having SEQ ID NO: 13 in the Sequence Listing Protein with amino acid sequence represented by IDNO:14 (mouse Siglec-15-Fc).

Example 2 Large-scale preparation of culture medium containing soluble mouse Siglec-15 protein using 293-F cells

Two types of expression plasmids (soluble mouse Siglec-15/pDONM and soluble mouse Siglec-15/phIgFc) obtained in Example 1 were prepared in an amount of about 5 mg, respectively. Also, when purifying a plasmid from large-scale cultured Escherichia coli, Invitrogen PureLink HiPure Plasmid Gigaprep Kit (manufactured by Invitrogen, Inc.) was used. The plasmid thus prepared was mixed with Opti-MEM (manufactured by Invitrogen, Inc.), the resulting mixture was filter-sterilized, 10 ml of transfection reagent 293fectin (manufactured by Invitrogen, Inc.) was added thereto, and the resulting mixture was placed in Incubate at room temperature for 20 minutes. The mixture was added to FreeStyle 293-F cells (manufactured by Invitrogen, Inc.) cultured in Erlenmeyer flasks to a cell density of 1.1 × 10 in FreeStyle 293 expression medium (manufactured by Invitrogen, Inc.). ⁶ cells/ml×5L (1L/flask, 5 bottles). After culturing the cells at 8.0% CO ₂ concentration at 37°C with shaking (125 rpm) for 96 hours (4 days), the culture solution was collected and centrifuged to prepare the culture supernatant. It is considered that in the culture supernatant thus prepared, a protein in which a V5 epitope tag and a 6×His tag have been added to the C-terminal side of the mouse Siglec-15 extracellular domain (mouse Siglec-15-His) is respectively expressed and a protein in which a human Fc tag has been added to the C-terminal side of the extracellular domain of mouse Siglec-15 (mouse Siglec-15-Fc).

Example 3 Purification of mouse Siglec-15-His

a) HisTrap HP column chromatography

Add 225ml of 10x buffer (500 mM Tris, 1.5 M NaCl, 200 mM imidazole, pH 8.0) to the culture medium of 293F cells expressing mouse Siglec-15-His prepared in Example 2, and stir evenly to obtain and filtered through a Sterivex-GV filter (manufactured by Millipore Co., Ltd.). At a flow rate of 2 ml/min, this culture solution was loaded onto three HisTrap HP 5 ml columns (manufactured by Amersham Biosciences, Inc.) in series, which had been previously treated with pyrogen remover PyroCLEAN (manufactured by ALerCHEK, Inc.), And washed with distilled water for injection. At a flow rate of 1 ml/min, the column was washed with 60 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl, and then 50 ml of 50 mM Tris-HCl buffer containing 300 mM NaCl and 500 mM imidazole ( pH 8.0), at a flow rate of 1ml/min, the protein adsorbed on the column was eluted. At 1 ml/fraction, the eluate was fractionated into Mini-sorp tubes (manufactured by Nunc, Inc.) to which 10 μl of 10% Tween 20 was previously added. Using a centrifugal membrane concentrator AmiconUltra-15 (manufactured by Millipore Co., Ltd.), about 20 ml of the solution containing the eluted protein obtained by combining the fractions (fraction 14-20) was concentrated to 2.5 ml, and then the concentrated The solution was loaded onto a PD-10 desalting column (manufactured by Amersham Biosciences, Inc.) equilibrated with phosphate-buffered saline (T-PBS) containing 0.01% Tween 20 in advance, and then eluted with T-PBS, thereby obtaining 3.5 ml For samples, the solvent was replaced with T-PBS.

b) Resource Q column chromatography

To 3.5 ml of the sample purified by HisTrap HP column chromatography whose solvent was replaced with T-PBS was added 22.5 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% CHAPS, and the resulting mixture was stirred. Then, the mixture was centrifuged at 4°C, 3,000 rpm for 30 minutes to remove the precipitate. After filtering the obtained supernatant through a Millex-GV filter (manufactured by Millipore Co., Ltd.), the filtrate was loaded at a flow rate of 1 ml/min into on a Resource Q 6 ml column (manufactured by Amersham Biosciences, Inc.). Thereafter, the buffer was used to wash the column at a flow rate of 1 ml/min, and the protein fraction not adsorbed to the column was collected. The protein adsorbed on the column was eluted with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% CHAPS and 1 M NaCl at a flow rate of 1 ml/min. 26.5 ml of the fraction not adsorbed on the column was concentrated to 2.0 ml with a centrifugal membrane concentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), and then the concentrate was centrifuged at 4° C., 3,000 rpm for 10 minutes, and Remove the precipitate. The supernatant after centrifugation was stored frozen at -80°C for later use. The above purification operation (HisTrap HP column chromatography and Resource Q column chromatography) was repeated twice.

c) Detection and purity analysis of purified mouse Siglec-15-His

SDS-polyacrylamide electrophoresis and silver staining under reducing conditions were performed using the samples prepared by the above-mentioned purification operations (HisTrap HP column chromatography and Resource Q column chromatography). That is, to 5 µl of each sample purified by each purification step, an equal amount of SDS-treated solution was added, and the resulting mixture was heat-treated at 95°C for 10 minutes. 3 μl of each heat-treated sample was used for SDS-polyacrylamide electrophoresis. An 8-25% polyacrylamide gradient gel (manufactured by Amersham Biosciences, Inc.) was used as an electrophoresis gel, and electrophoresis was performed using a PhastSystem (manufactured by Amersham Biosciences, Inc.). In addition, rainbow molecular weight marker (manufactured by Amersham Biosciences, Inc.) was used as the molecular weight marker. After electrophoresis, silver staining was performed using PhastGel Silver Kit (manufactured by Amersham Biosciences, Inc.) and PhastSystem. The result is shown in Figure 1. It was confirmed that a protein with a molecular weight of about 35 kDa (mouse Siglec-15-His) was efficiently purified and concentrated in the protein fraction not adsorbed on the Resource Q column.

Electrophoresis was performed under the same conditions except that ECL DualVue Western Blotting Marker (manufactured by Amersham Biosciences, Inc.) was used as a molecular weight marker. The protein in the gel was transferred (blotted) onto a PVDF membrane (Hybond-P, manufactured by Amersham Biosciences, Inc.) using PhastTransfer Semi-Dry Transfer Kit (manufactured by Amersham Biosciences, Inc.) and PhastSystem. The PVDF membrane was gently shaken at room temperature for 1 hour in 10 ml of blocking agent (BlockAce, produced by Snow Brand Milk Products, Co., Ltd.) containing 0.1% Tween 20. 10 μl of S-protein HRP (manufactured by Amersham Biosciences, Inc.) and 10 μl of anti-V5-HRP antibody (monoclonal antibody against Pk-TAG-HRP, produced by Acris Antibodies) were added to the blocking solution, and the membrane in the solution was Shake gently for another 1 hour at room temperature. Wash the PVDF membrane 4 times by gently shaking for 5 min in 50 ml of phosphate-buffered saline (PBS) containing 0.01% Tween 20. After washing, the PVDF membrane was treated according to the method attached to the ECL detection kit (produced by Amersham Biosciences, Inc.) to develop the color of the protein bands, and the ECL Mini-Camera (produced by Amersham Biosciences, Inc.) and polarizing film (Polapan 3200B, produced by Polaroid, Inc.) to detect color development. The result is shown in Figure 2. It was also confirmed from this result that in the protein fraction not adsorbed to the Resource Q column, the protein (mouse Siglec-15-His) having a molecular weight of about 35 kDa and reacting with the anti-V5-HRP antibody was efficiently purified and concentrated.

d) Determination of protein purity of purified mouse Siglec-15-His

For the purified mouse Siglec-15-His (the protein fraction not adsorbed to the Resource Q column), using bovine serum albumin as a standard, the protein was measured with the DC protein assay kit (manufactured by Bio-Rad Laboratories, Inc.) concentration. As shown in Table 1, a total of 1.66 mg of purified mouse Siglec-15-His protein was obtained by performing two purification operations.

Table 1

.

Embodiment 4 Purification of mouse Siglec-15-Fc

a) HiTrap protein A column chromatography

Filter 1.8 L of the culture solution of 293F cells expressing mouse Siglec-15-Fc prepared in Example 2 through a Sterivex-GV filter (manufactured by Millipore Co., Ltd.), and then filter the filtrate at a flow rate of 5 ml/min It was loaded onto a HiTrap protein A 5 ml column (manufactured by Amersham Biosciences, Inc.) pre-equilibrated with Dulbecco's PBS (D-PBS, produced by Invitrogen, Inc.). After the column was washed with D-PBS at a flow rate of 5 ml/min, the protein adsorbed on the column was eluted with 50 ml of 0.1 M sodium citrate buffer (pH 3.0) at a flow rate of 5 ml/min. At 5 ml/fraction, the eluate was fractionated into mini-sorp tubes (manufactured by Nunc, Inc.), and then 1.3 ml of 1 M Tris was immediately added to neutralize the eluate. Using a centrifugal membrane concentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), the solution obtained by combining the fractions (1 and 2) in which the eluted protein was detected was concentrated to 2.5 ml, and then the concentrated The solution was loaded onto a PD-10 desalting column (manufactured by Amersham Biosciences, Inc.) equilibrated with Otsuka saline for injection (TO-SS, produced by Otsuka Pharmaceutical Co., Ltd.) containing 0.01% Tween 20, followed by TO-SS -SS eluted, thus obtaining a 3.5ml sample whose solvent was replaced by TO-SS. The samples were stored frozen at -80°C until use. Using 2.9L of 293F cell culture medium, repeat the same purification operation once more.

b) Detection and purity determination of purified mouse Siglec-15-Fc

Using the sample prepared by the above purification method, SDS-polyacrylamide electrophoresis and silver staining under reducing conditions were performed. That is, to 5 µl of each sample purified by each purification step, an equal amount of SDS-treated solution was added, and the resulting mixture was heated at 95°C for 10 minutes. Each heat-treated sample was diluted to 1/300 or 1/900 with a half-concentration SDS-treatment solution, and then 0.3 μl of the sample was used for SDS-polyacrylamide electrophoresis. Electrophoresis and silver staining were performed in the same manner as in the measurement of the purity of mouse Siglec-15-His described in c) of Example 3. The results are shown in Fig. 3 together with those of a small-scale examination of the preliminary purification conditions (the pH of the applied culture broth was 8.9 or 7.0). It was confirmed that in the protein fraction eluted from the HiTrap Protein A column, a protein with a molecular weight of about 55 kDa (mouse Siglec-15-Fc) was efficiently purified and concentrated.

c) Determination of protein purity of purified mouse Siglec-15-Fc

For purified mouse Siglec-15-Fc (protein fraction eluted from a PD-10 desalting column), using bovine serum albumin as a standard, DC protein assay kit (manufactured by Bio-Rad Laboratories, Inc.) Determine protein concentration. As shown in Table 2, a total of 92 mg of purified mouse Siglec-15-Fc protein was obtained through two purification operations.

Table 2

.

Example 5 Establishment of hybridoma producing rat anti-mouse Siglec-15 monoclonal antibody

a) Antigen preparation

The mouse Siglec-15-His protein produced in Example 3 was prepared at 100 μg/0.5 ml, an equal amount of adjuvant was added thereto, and an emulsion was made using a glass syringe. As an adjuvant, Freund's complete adjuvant (manufactured by FCA, Difco Laboratories, Inc.) was used only for the first immunization, and Freund's incomplete adjuvant (FICA, manufactured by Difco Laboratories, Inc.) was used for the second and subsequent immunizations.

b) Immunization of rats

Four rats (Wistar, female, 6 weeks old, purchased from CLEA Japan, Inc.) were used as immunized animals. Using a 27G injection needle, the emulsion obtained in a) was injected subcutaneously and intradermally so that the amount of the antigen would be 50 µg/rat. After the first immunization, immunization was carried out every 7 days for a total of 4 times. Seven days after the fourth immunization, a small amount (200 µl) of blood was collected from the tail vein to prepare antiserum. In order to confirm the antibody titer of the antiserum, ELISA was performed in which the mouse Siglec-15-His protein that had been used as an antigen, the mouse Siglec-15-Fc protein prepared in Example 4, or bovine serum albumin (BSA ), which are each solid-phased. As a result, reactivity with mouse Siglec-15-His protein and mouse Siglec-15-Fc protein was observed in all four rats (rat numbers 1 to 4). On the other hand, no reactivity with BSA was observed. From these results, it was confirmed that the antibody titer in the sera of each immunized rat was elevated, so the cell fusion operation was performed on rat No. 2 showing the highest antibody titer.

c) cell fusion

Cell fusion was performed according to an ordinary method for fusing mouse (rat) spleen cells and myeloma cells. Whole blood was collected from the heart of the rat under ether anesthesia, and the rat was euthanized, and then the spleen was removed. Harvested spleen cells were fused with P3X63Ag8.653 cells (ATCC CRL 1580) (mouse myeloma cells) using polyethylene glycol (PEG). The obtained cells were seeded in a 96-well plate, and a medium (HAT selection medium) containing hypoxanthine (H), aminopterin (A) and thymidine (T) was added thereto, and the cells were cultured for 7~ 10 days. Culture supernatants were collected from 61 wells in which survival of hybridomas obtained by cell fusion was confirmed. Then, using the mouse Siglec-15-His protein that had been used as an antigen, the mouse Siglec-15-Fc protein prepared in Example 4, or BSA (each of which was immobilized), the antibody titer was evaluated by ELISA, and screened Hybridoma producing anti-mouse Siglec-15 monoclonal antibody. Based on the screening results, 12 wells exhibiting high antibody titers were selected, and hybridomas contained in the wells were subjected to cloning operations.

d) Cloning of hybridomas

The hybridomas thus selected were subjected to a first cloning step by the limiting dilution method. After limiting dilution, the hybridoma was cultured for 2 weeks, and the antibody titer in the culture supernatant was confirmed by ELISA using the mouse Siglec-15-Fc protein prepared in Example 4 or BSA (each of which was immobilized) . For the 11 clones confirmed as positive clones, the second cloning step was carried out (in the same manner as the first cloning step), whereby 10 hybridomas producing anti-mouse Siglec-15 monoclonal antibody were finally established. clones (#1A1, #3A1, #8A1, #24A1, #32A1, #34A1, #39A1, #40A1, #41B1, #61A1). In addition, on August 28, 2008, the hybridoma #32A1 was deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology, located at: Central6, 1- 1-1 Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), and has obtained accession number FERM BP-10999 under the name of anti-Siglec-15 hybridoma #32A1.

Example 6 Preparation of Rat Anti-Mouse Siglec-15 Monoclonal Antibody

a) Preparation of nude mouse ascites

The hybridoma established in Example 5 was cultured using TIL medium I (manufactured by Immuno-biological Laboratories Co., Ltd.) medium containing 10% FCS. The subculture of the cells was carried out by the following method, wherein the time point at which the cells grew to about 5 x 10 ⁵ cells/ml was used as an index, and the culture solution was diluted to about 1/4 every 2 to 3 days. Each of the hybridomas thus cultured was implanted intraperitoneally into nude mice previously intraperitoneally administered (0.2 ml/mouse) pristane at 1 x 10 ⁷ cells/mouse. In implantation, 3 nude mice were used for each of the 10 hybridoma clones. After implantation, when sufficient accumulation of ascites was observed, collect the ascites, combine it with ascites collected from two other mice implanted with the same hybridoma, measure the amount of the thus combined ascites, and cryopreserve the ascites until the antibody purification. The amount of ascitic fluid collected for each hybridoma is summarized in Table 3.

table 3

.

b) Purification of antibodies

The total amount of collected ascites was subjected to IgG purification using a 20 ml protein G column (manufactured by GE Healthcare, Co., Ltd.). Purified IgG was assayed for purity by gel filtration analysis (Superdex 200 column chromatography), and a fraction of antibodies were centrifuged membrane concentrated. That is, in the purified anti-mouse Siglec-15 monoclonal antibody, the antibody was centrifuged at 3,000 rpm at 4°C for 30 to 60 minutes using a centrifugal membrane concentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.). , Concentrate the class 9 antibodies except #24A1 antibody to about 1/6-1/8 of the original volume. Next, using bovine serum albumin (BSA) as a standard, for the #24A1 antibody and other concentrated class 9 antibodies, the protein concentration was measured with a DC protein assay kit (manufactured by Bio-Rad Laboratories, Inc.). Through the above operations, anti-mouse Siglec-15 monoclonal antibody was prepared.

Example 7 Evaluation of the Binding Properties of Rat Anti-Mouse Siglec-15 Monoclonal Antibody to Mouse Siglec-15 Protein

The binding property of rat anti-mouse Siglec-15 monoclonal antibody to mouse Siglec-15 protein was evaluated by ELISA method. With 0.1M sodium carbonate buffer (pH 9.5), the mouse Siglec-15-Fc protein prepared in Example 4 was diluted to 5 μg/ml, and the resulting solution was added to a 96-well plate (Nalge Nunc International, Inc., catalog number 430341). After leaving the plate at room temperature for 1 hour, the solution was removed, and a washing buffer (phosphate-buffered saline containing 0.05% Tween 20) was added at 300 μl/well, and then removed. After this washing operation was performed once more, 200 µl/well of phosphate-buffered saline containing 25% BlockAce (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) was added, and the plate was left to stand at room temperature for 1 hour to thereby block. The liquid was removed and the plate was washed twice with 300 μl/well of wash buffer. Then, each of the rat anti-mouse Siglec-15 monoclonal antibodies prepared in Example 6 or the rat control IgG (manufactured by R&D Systems, Inc.) was diluted to a final concentration of 1.28 to 20,000 ng/ml (5-fold dilution series), and the resulting diluted antibody solution was added to the plate at 100 μl/well. After the plate was left at room temperature for 1 hour, the liquid was removed, and the plate was washed 3 times with 300 µl/well of washing buffer. Next, HRP (horseradish peroxidase)-labeled goat anti-rat IgG antibody (manufactured by Beckman Coulter, Inc.) diluted to 1,000-fold with ELISA buffer was added at 100 μl/well, and the plate was incubated at room temperature. Leave for 1 hour. The solution was removed, washed 3 times with 300 µl/well of washing buffer, and then color development was performed using a color development kit for peroxidase (manufactured by SumitomoBakelite Co., Ltd.) according to the instructions attached to the kit. After color development, the absorbance at 492 nm was measured with a microplate reader (manufactured by Nihon Molecular Devices Corporation). The results confirmed that all of the 10 rat anti-mouse Siglec-15 monoclonal antibody test samples examined bound to mouse Siglec-15 protein in an antibody concentration-dependent manner ( FIG. 4 ). On the other hand, in the case of rat control IgG, no binding to mouse Siglec-15 protein was observed.

Example 8 Preparation of mouse bone marrow non-adherent cells

Femurs and tibiae were removed from 5–8-week-old male ddY mice, and soft tissues were removed. Cut both ends of the femur or tibia, inject D-PBS with a syringe with a 25-gauge needle to push out the bone marrow cells, and collect them in a centrifuge tube. Centrifuge at 100 g for 5 minutes at room temperature and remove the supernatant. 1 ml of hemolysis buffer (erythrocyte lysis buffer, manufactured by Sigma Co., Ltd.) was added to the obtained cell pellet for suspension, and the obtained suspension was left at room temperature for 5 minutes. 20 ml of D-PBS was added thereto, the suspension was centrifuged at 100 g for 5 minutes at room temperature, and the supernatant was removed. To the obtained cell pellet, 10 ml of MEM-α medium (produced by Invitrogen, Inc.) containing 5 ng/ml M-CSF (produced by R&D Systems, Inc.) and 10% fetal bovine serum (FBS) was added to mix. hanging. Then, the resulting suspension was passed through a cell strainer (40 μm nylon, produced by BD Falcon) to remove aggregates. The resulting cells were transferred to a 75 cm ² T-flask (for attachment of adherent cells) and cultured overnight in a CO ₂ incubator. After overnight culture, cells not attached to the T-flask were collected and used as mouse bone marrow non-adherent cells.

Example 9 Evaluation of Biological Activity of Rat Anti-Mouse Siglec-15 Monoclonal Antibody Based on Mouse Osteoclast Formation Assay

Using all of the 10 anti-mouse Siglec-15 monoclonal antibody test samples prepared in Example 6, the effect on osteoclast differentiation of mouse bone marrow non-adherent cells was examined. In the α-MEM medium containing 10% FBS and 10 ng/ml M-CSF (manufactured by R&D Systems, Inc.), the mouse bone marrow non-adherent cells prepared by the method of Example 8 were made into 1.5×10 ⁵ cells/ml, and inoculate 200 μl of the obtained cell preparation into each well of a 96-well plate, and culture the cells in a CO ₂ incubator for 2 days. The old culture solution in the 96-well plate was removed, and 100 μl of MEM-α medium containing 10% FBS to which human RANKL (manufactured by RANKL, Peprotech, Inc.) had been added was added to each well and M-CSF, the final concentrations were 20 ng/ml and 10 ng/ml, respectively. Each of the rat anti-mouse Siglec-15 monoclonal antibodies prepared in Example 6, a commercially available rat control IgG (purified large Rat IgG, manufactured by R&D Systems, Inc.) samples obtained by removing sodium azide, or rabbit anti-mouse Siglec-15 polyclonal antibody (No. 3) prepared separately, and cultured cells in a CO ₂ incubator Another 3 days. In addition, polyclonal antibody (No. 3) is an antibody that has been confirmed to inhibit osteoclast formation in the experimental system described in this example. After the end of the culture, the activity of tartrate-resistant acid phosphatase (TRAP) of the formed osteoclasts was measured by the following method. The culture medium in each well of the 96-well plate was removed by aspiration, and 50 μl of 50 mM sodium citrate buffer (pH 6.1) containing 1% Triton X-100 was added to each well. Then, shake the plate on a plate shaker for 5 minutes to lyse the cells. Add 50 μl of substrate solution (50 mM sodium citrate buffer (pH 6.1) containing 5 mg/ml p-nitrophenyl phosphate and 0.46% sodium tartrate) to each well, and incubate the plate at room temperature. Incubate for 5 minutes. After incubation, 50 µl of 1N sodium hydroxide solution was added to each well of the 96-well plate to stop the enzyme reaction. After the enzymatic reaction was stopped, the absorbance at 405 nm of each well was measured, and the resulting measurement was used as an index of TRAP activity. The results are shown in Figures 5 and 6. In the case of the commercially available rat control IgG, no significant inhibition of TRAP activity was observed. On the other hand, in the case of adding #32A1 antibody in the range of 32 ng/ml to 1000 ng/ml, and in the case of adding #8A1 antibody and affinity-purified rabbit polyclonal in the range of 63 ng/ml to 1000 ng/ml In the case of No. 3 antibody, significant inhibition of TRAP activity was observed. Also in the case of #3A1 antibody, #34A1 antibody and #39A1 antibody, dose-dependent inhibition of TRAP activity was observed at a relatively high concentration of 500 ng/ml or more. Inhibition of mouse osteoclast formation by other antibodies was not observed. From the above results, among the prepared rat anti-mouse Siglec-15 monoclonal antibodies, antibodies that strongly inhibit mouse osteoclast formation (osteoclast differentiation and maturation) were found. In addition, as common properties of the #3A1 antibody, #8A1 antibody, #32A1 antibody, #34A1 antibody, and #39A1 antibody, activity to inhibit osteoclast formation at a concentration of 1000 ng/ml (i.e., 1 μg/ml) or less can be exemplified .

Preparation of embodiment 10 soluble human Siglec-15 protein expression construct

The partial nucleotide sequence encoding the extracellular domain of human Siglec-15 protein is represented by SEQ ID NO: 15 in the sequence listing, and its amino acid sequence is represented by SEQ ID NO: 16 in the sequence listing. By utilizing such a partial sequence, soluble human Siglec-15 protein can be produced in the culture supernatant of animal cells or the like.

a) Amplification of soluble human Siglec-15 gene by PCR

According to a conventional method, an oligonucleotide having the sequence 5'-ggggacaagt ttgtacaaaa aagcaggcttcaccATGGAA AAGTCCATCT GGCTGC-3' (hSiglec-15-ECD-F: SEQ ID NO: 17 in the sequence listing) and an oligonucleotide having the sequence 5'-ggggaccact ttgtacaaga were synthesized. The oligonucleotide of aagctgggtc CCCGCTGGCG CCATGGAAGCGG-3' (hSiglec-15-ECD-R: SEQ ID NO: 18 in the Sequence Listing) was used as a primer for amplifying the extracellular domain of human Siglec-15 by PCR. In addition, these primers were designed as amplification primers for the production of gateway entry clones to add the attB1 sequence in hSiglec-15-ECD-F and the attB2 sequence in hSiglec-15-ECD-R. PCR was carried out using this primer set and a polynucleotide containing the open reading frame sequence of human Siglec-15 as a template according to a conventional method. The resulting PCR reaction solution was purified using PureLink PCR Purification Kit (manufactured by Invitrogen, Inc.).

b) Preparation of entry clones by Gateway BP reaction

An entry clone in which human Siglec-15 extracellular domain cDNA was integrated by Gateway technology (Invitrogen, Inc.) using a λ bacteriophage site-specific recombination system was prepared by the following method. First, between the PCR product having the attB sequence prepared in a) at both ends and pDNOR221 (manufactured by Invitrogen, Inc., which is a donor vector having attP sequence), a BP reaction was performed using BP Clonase. Using this reaction solution, Escherichia coli TOP10 was transformed, colony PCR was performed for the drug-resistant clone, and the size of the insert was confirmed. Then, for clones confirmed to have an insert of the correct size, sequence analysis of the total DNA sequence of the insert was performed. As a result, an incoming clone completely identical to the target nucleotide sequence (SEQ ID NO: 15 in the Sequence Listing) encoding the extracellular domain of the human Siglec-15 protein was obtained.

c) Preparation of expression clones by Gateway LR reaction

An expression clone was prepared in which human Siglec-15 extracellular domain cDNA was integrated in the entry clone by Gateway technology (Invitrogen, Inc.) using a λ bacteriophage site-specific recombination system. The entry clone prepared in b) contains an insert with attL sequences at both ends. Between this entry clone and the two types of destination vectors with attR sequences, an LR reaction was performed using LR Clonase. Furthermore, as the destination vector, two types of destination vectors were used: pDONM designed to add a V5 epitope tag and 6×His tag to the C-terminus of the insert, and phIgFc designed to add a human Fc tag to the C-terminus of the insert. By using the reaction solution obtained from the LR reaction, transform Escherichia coli TOP10, and perform sequence analysis on the resulting drug-resistant clone to confirm whether correct recombination occurs.

As a result of the sequence analysis, expression clones (soluble human Siglec-15/pDONM and soluble human Siglec-15/phIgFc) in which correct recombination of pDONM and phIgFc occurred were respectively obtained. By transfecting soluble human Siglec-15/pDONM into animal cells, etc., the mRNA having the base sequence represented by SEQ ID NO: 19 in the sequence listing is transcribed, and is translated into having the SEQ ID NO in the sequence listing: The protein with the amino acid sequence represented by 20 (human Siglec-15-His). In addition, by transfecting soluble human Siglec-15/phIgFc into animal cells and the like, mRNA having a base sequence represented by SEQ ID NO: 21 in the Sequence Listing is transcribed and translated into one having SEQ ID NO in the Sequence Listing : A protein (human Siglec-15-Fc) with an amino acid sequence represented by 22.

Example 11 Large-scale preparation of culture medium containing soluble human Siglec-15 protein using 293-F cells

a) Preparation of culture medium containing human Siglec-15-His

The soluble human Siglec-15/pDONM obtained in Example 10 was prepared in an amount of about 25 mg. Also, when purifying plasmids from large-scale cultured E. coli, Invitrogen PureLink HiPure PlasmidGigaprep Kit (manufactured by Invitrogen, Inc.) was used. The plasmid thus prepared was mixed with Opti-MEM (manufactured by Invitrogen, Inc.), 50 ml of transfection reagent 293fectin (manufactured by Invitrogen, Inc.) was added thereto, and the resulting mixture was incubated at room temperature for 20 minutes. This mixture was added to FreeStyle 293-F cells (manufactured by Invitrogen, Inc.), which were cultured in FreeStyle 293 expression medium (manufactured by Invitrogen, Inc.) containing 1% penicillin-streptomycin, so that the cells Densities of ^1.0-3.4 x 106 cells/ml were achieved using a 25 L bioprocess culture unit (WAVE bioreactor). After culturing the cells for 96 hours (4 days) at 37°C with shaking (30 rpm) at a concentration of 6-12% CO ₂ , the culture solution was collected and centrifuged to prepare the culture supernatant. It was observed that in the culture supernatant thus prepared, a protein in which a V5 epitope tag and a 6xHis tag had been added to the C-terminal side of the extracellular domain of human Siglec-15 (human Siglec-15-His) was expressed.

b) Preparation of culture medium containing human Siglec-15-Fc

The soluble human Siglec-15/phIgFc obtained in Example 10 was prepared in an amount of about 5 mg. Also, when purifying plasmids from large-scale cultured E. coli, Invitrogen PureLink HiPure PlasmidGigaprep Kit (manufactured by Invitrogen, Inc.) was used. The plasmid thus prepared was mixed with Opti-MEM (manufactured by Invitrogen, Inc.), followed by filter sterilization. Then, 10 ml of transfection reagent 293fectin (manufactured by Invitrogen, Inc.) was added thereto, and the resulting mixture was incubated at room temperature for 20 minutes. This mixture was added to FreeStyle 293-F cells (manufactured by Invitrogen, Inc.) cultured in Erlenmeyer flasks so that the cell density in FreeStyle 293 Expression Medium (manufactured by Invitrogen, Inc.) reached 1.0-3.0 x 10 ⁶ cells/ml x 5 L (1 L/flask x 5 flasks). After culturing the cells at 8.0% CO ₂ concentration at 37°C with shaking (125 rpm) for 96 hours (4 days), the culture solution was collected and centrifuged to prepare the culture supernatant. It was observed that in the culture supernatant thus prepared, a protein in which a human Fc tag had been added to the C-terminal side of the extracellular domain of human Siglec-15 (human Siglec-15-Fc) was expressed.

Example 12 Purification of Soluble Human Siglec-15 Protein

a) Purification of soluble human Siglec-15-His

a-i) HisTrap HP column chromatography

1350 ml of 10x buffer (500 mM Tris, 1.5 M NaCl, 200 mM imidazole, pH 8.0) was added to 12 L of culture medium of 293F cells expressing human Siglec-15-His prepared in a) of Example 11, The resulting mixture was stirred uniformly, and filtered with a MilliPak-60 filter (manufactured by Millipore Co., Ltd.). At a flow rate of 10 ml/min, this culture solution was loaded onto a Ni-Sepharose HP (manufactured by Amersham Biosciences, Inc.) 100 ml column washed with pure water (Milli Q water) in advance. After washing the column with 400 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl at a flow rate of 8 mL/min, wash the column with 200 mL of 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 500 mM imidazole at 2.5 The protein adsorbed on the column was eluted at a flow rate of mL/min, and the eluate was fractionated into mini-sorp tubes (manufactured by Nunc, Inc.). To prevent protein precipitation, 8 mL of 5M NaCl solution was added to about 40 mL of the fraction containing the eluted protein, followed by stirring, and then the resulting mixture was concentrated using a centrifugal membrane concentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.) to about 20 ml. Insoluble matter generated upon concentration was removed by centrifugation at 3000 rpm at 4°C for 30 minutes, and 2.5 mL of the resulting supernatant was loaded onto PD-10 pre-equilibrated with phosphate-buffered saline (N-PBS) containing 1 M NaCl. A desalting column (manufactured by Amersham Biosciences, Inc.), followed by elution with N-PBS, thereby obtaining a 3.5 mL sample in which the solvent was replaced with N-PBS. This operation was repeated another 7 times to obtain about 28 mL of a partially purified human Siglec-15-His solution.

a-ii) Resource Q column chromatography

In 50 mM Tris-HCl buffer (pH 7.5) (1 L, 3 times) containing 0.1% CHAPS, 12 ml of the sample, which had been purified by Ni-Sepharose HP column chromatography, was dialyzed overnight at 4°C, and The solvent thereof had been replaced with N-PBS, and the obtained dialysate was centrifuged at 4° C., 3,000 rpm for 30 minutes to remove precipitates. After filtering the obtained supernatant with a Millex-GV filter (manufactured by Millipore Co., Ltd.), at a flow rate of 1 ml/min, the filtrate was loaded into a solution previously filled with 50 mM Tris-HCl buffer (pH 7. 5) Equilibrated Resource Q 6 ml column (manufactured by Amersham Biosciences, Inc.). Thereafter, the column was washed at a flow rate of 1 ml/min, the column was washed with this buffer, and the protein fraction not adsorbed on the column was collected. At a flow rate of 1 ml/min, the protein adsorbed on the column was eluted with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1% CHAPS and 1 M NaCl. After concentrating 26.5 ml of the fraction not adsorbed on the column to 3.0 ml with a centrifugal membrane concentrator Amicon Ultra-15 (manufactured by Millipore Co., Ltd.), the concentrate was centrifuged at 4°C, 3,000 rpm for 10 minutes, and the precipitate was removed . 2.5 mL of the obtained supernatant was loaded onto a PD-10 desalting column (manufactured by Amersham Biosciences, Inc.) equilibrated in advance with phosphate-buffered saline (pH 7.0, A-PBS) containing 50 mM arginine hydrochloride, and then washed with A- PBS was eluted, thereby obtaining a 3.5 mL sample in which the solvent was replaced with A-PBS. To prevent the precipitation of soluble human Siglec-15-His, arginine hydrochloride was added to the prepared sample solvent. The supernatant after centrifugation was stored frozen at -80°C for future use. The above purification operation (Resource Q column chromatography) was repeated twice.

a-iii) Detection and purity determination of purified human Siglec-15-His

Using the samples prepared by the above purification operations (Ni-Sepharose HP column chromatography and Resource Q column chromatography), SDS-polyacrylamide electrophoresis and silver staining under reducing conditions were performed. That is, to 5 µl of each sample purified by the respective purification steps, an equal amount of SDS-treated solution was added, and the resulting mixture was heat-treated at 95°C for 10 minutes. 0.3 μl of each heat-treated sample was used for SDS-polyacrylamide electrophoresis. Electrophoresis was performed in the same manner as in Example 3, except that Rainbow Molecular Weight Marker (manufactured by Amersham Biosciences, Inc.) was used as the molecular weight marker. After electrophoresis, silver staining was performed using PhastGel Silver Kit (manufactured by Amersham Biosciences, Inc.) and PhastSystem. The result is shown in Figure 7. It was shown that in the protein fraction not adsorbed on the Resource Q column, a protein (human Siglec-15-His) with a molecular weight of about 35 kDa was efficiently purified and concentrated.

a-iv) Determination of protein concentration of purified human Siglec-15-His

For purified human Siglec-15-His (the protein fraction not adsorbed on the Resource Q column), using bovine serum albumin as a standard, the protein was measured with a DC protein assay kit (manufactured by Bio-Rad Laboratories, Inc.) concentration. Through two purification operations, a total of 1.66 mg of purified human Siglec-15-His was obtained.

b) Purification of soluble human Siglec-15-Fc

b-i) HiTrap protein A column chromatography

1.5 L of the culture solution of 293F cells expressing human Siglec-15-Fc prepared in b) of Example 11 was filtered through a Sterivex-GV filter (manufactured by Millipore Co., Ltd.), and then at a flow rate of 5 ml/min , and the filtrate was loaded onto a HiTrap Protein A 5 ml column (manufactured by Amersham Biosciences, Inc.) equilibrated in advance with Dulbecco's PBS (D-PBS, produced by Invitrogen, Inc.). After washing the column with 70 ml of D-PBS at a flow rate of 5 ml/min, the protein adsorbed on the column was eluted with 24 ml of 0.1 M sodium citrate buffer (pH 3.0) at a flow rate of 1.2 ml/min. At 1.2 ml/fraction, the eluate was fractionated into mini-sorp tubes (manufactured by Nunc, Inc.), and then 0.31 ml of 1MTris was immediately added thereto to neutralize the eluate. A 2.5 ml aliquot of the solution (approximately 7.5 ml) obtained by pooling the eluted protein fractions (fractions 5-9) was loaded into phosphate-buffered saline (pH 7.0) containing 50 mM arginine hydrochloride. , A-PBS) equilibrated PD-10 desalting column (manufactured by Amersham Biosciences, Inc.), and then eluted with A-PBS, thereby obtaining a 3.5 ml sample in which the solvent was replaced with A-PBS. This operation was repeated 2 times. To prevent the precipitation of soluble human Siglec-15-Fc, arginine hydrochloride was added to the solvent. The 2.5ml remaining solution of the eluted protein fractions (fractions 5-9) was loaded onto a PD-10 desalting column (Amersham Biosciences, Inc.), and then eluted with N-PBS, thereby obtaining a 3.5 ml sample in which the solvent was replaced with N-PBS. In order to prevent the precipitation of soluble human Siglec-15-Fc, NaCl was added to the solvent of the prepared sample without adding an amino group-containing compound such as arginine. The samples prepared by the above operations were stored frozen at -80°C for future use.

b-ii) Detection and purity determination of purified human Siglec-15-Fc

Using the sample prepared by the above purification operation, SDS-polyacrylamide electrophoresis and silver staining under reducing conditions were performed. That is, to 5 µl of each sample purified by the respective purification steps, an equal amount of SDS-treated solution was added, and the resulting mixture was heated at 95°C for 10 minutes. Each heat-treated sample was diluted to 1/100 or 1/300 with a half-concentration SDS-treatment solution, and 0.3 µl of the resulting sample was used for SDS-polyacrylamide electrophoresis. Electrophoresis and silver staining were performed in the same manner as in the determination of the purity of human Siglec-15-His described in a-iii). The result is shown in Figure 8. It was shown that in the protein fraction eluted from the HiTrap protein A column, a protein (human Siglec-15-Fc) with a molecular weight of about 55 kDa was efficiently purified and concentrated.

b-iii) Determination of protein concentration of purified human Siglec-15-Fc

For purified human Siglec-15-Fc (protein fraction eluted from a PD-10 desalting column), bovine serum albumin was used as a standard and measured with a DC protein assay kit (manufactured by Bio-Rad Laboratories, Inc.) protein concentration. As shown in Table 4, a total of 25.2 mg of purified human Siglec-15-Fc was obtained through two purification operations.

Table 4

.

Example 13 Evaluation of the Binding Properties of Rat Anti-Mouse Siglec-15 Monoclonal Antibody to Human Siglec-15 Protein

The binding properties of rat anti-mouse Siglec-15 monoclonal antibody to human Siglec-15 protein were evaluated by ELISA method. The human Siglec-15-Fc protein prepared in b) of Example 12 (replaced with A-PBS) was diluted to 5 μg/ml with 0.1 M sodium carbonate buffer (pH 9.5), and the resulting solution was diluted in 100 μl /well was added to a 96-well plate (manufactured by Nalge Nunc International, Inc., cat. no. 430341). After leaving the plate at room temperature for 1 hour, the solution was removed, and a washing buffer (phosphate-buffered saline containing 0.05% Tween 20) was added at 300 μl/well, and then removed. After this washing operation was performed once more, phosphate-buffered saline containing 25% BlockAce (manufactured by Dainippon Sumitomo Pharma Co., Ltd.) was added at 200 μl/well, and the plate was left to stand at room temperature for 1 hour to thereby block. The liquid was removed and the plate was washed twice with 300 μl/well of wash buffer. Then, each of the rat anti-mouse Siglec-15 monoclonal antibodies prepared in Example 6 or the rat control IgG (manufactured by R&D Systems, Inc.) was diluted to a final concentration of 1.28 to 20,000 ng/ml (5-fold dilution series), and the resulting diluted antibody solution was added to the plate at 100 μl/well. After the plate was left at room temperature for 1 hour, the liquid was removed and the plate was washed 3 times with 300 μl/well of washing buffer. Subsequently, HRP (horseradish peroxidase)-labeled goat anti-rat IgG antibody (manufactured by Beckman Coulter, Inc.) diluted to 1,000-fold with ELISA buffer was added at 100 μl/well, and the plate was left at room temperature 1 hour. The liquid was removed, the plate was washed 3 times with 300 µl/well of washing buffer, and then, color development was carried out by using a color development kit for peroxidase (manufactured by Sumitomo Bakelite Co., Ltd.) according to the instructions attached to the kit . After color development, the absorbance at 492 nm was measured with a microplate reader (manufactured by Nihon Molecular Devices Corporation). The results confirmed that all 10 test samples of rat anti-mouse Siglec-15 monoclonal antibody examined bound human Siglec-15 protein in an antibody concentration-dependent manner ( FIG. 9 ). Specifically, 5 test samples (i.e. #1A1, #3A1, #24A1, #32A1 and #61A1) had higher binding activities, and 3 test samples (i.e. #8A1, #34A1 and #39A1) had relatively higher binding activities. Low. On the other hand, in the case of rat control IgG, no binding to human Siglec-15 protein was observed. The above results show that the rat anti-mouse Siglec-15 monoclonal antibody prepared in Example 6 not only binds to mouse Siglec-15, but also binds to human Siglec-15. In addition, some antibodies were found to strongly bind to human Siglec-15 .

Example 14 Effect of adding rat anti-mouse Siglec-15 monoclonal antibody on cell fusion and bone resorption activity of normal human osteoclast precursor cells (in vitro biological activity evaluation)

Since it was demonstrated in Example 13 that rat anti-mouse Siglec-15 monoclonal antibodies also bind to human Siglec-15, the effects of these antibodies on human osteoclastogenesis and bone resorption activities were examined.

a) Effect of adding rat anti-mouse Siglec-15 monoclonal antibody on cell fusion of osteoclasts from normal human osteoclast precursor cells (TRAP staining)

Normal human osteoclast precursor cells (normal human natural osteoclast precursor cells, purchased from Sanko Junyaku Co., Ltd., catalog number ^2T- 110) seeded in 96-well plate. Minimal Essential Medium for Osteoclast Precursor Cells (OPBM, purchased from Sanko Junyaku Co., Ltd., cat. No. PT-8201) as the medium, and the OPGM supplement set contains fetal bovine serum (final concentration 10%), human RANKL (final concentration 66 ng/ml), human M-CSF (final concentration 33 ng/ml) and the like. Each of the rat anti-mouse Siglec-15 monoclonal antibodies prepared in Example 6 or rat control IgG (manufactured by R&D Systems, Inc.) was added to the obtained culture supernatant to obtain a final concentration of 30 μg/ ml, and culture the cells for 4 days in a _CO incubator. After culturing, the supernatant was removed, and 10% neutral formalin was added to fix the cells. After the cells were fixed, the cells were washed twice with distilled water, and TRAP staining solution (0.27 mM naphthol AS-MX phosphate (manufactured by Sigma Co., Ltd.), 1.6 mM fast red purple LB salt (Sigma Co. , Ltd.), 1% dimethylformamide, 50 mM sodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)), and reacted at room temperature for 5 minutes. After the reaction, the cells were washed twice with distilled water, and then observed by microscopy ( FIG. 10 ). As a result, the addition of the #32A1 antibody almost completely inhibited the formation of giant osteoclasts resulting from high cell fusion. Furthermore, in the case of #41B1 antibody, the formation of giant osteoclasts resulting from high cell fusion was also significantly inhibited. On the other hand, this significant inhibition of osteoclast cell fusion was not observed with other rat anti-mouse Siglec-15 monoclonal antibodies (#1A1 antibody and other antibodies) and rat control IgG. In this way, it was confirmed that multinucleation and cell fusion of TRAP-positive osteoclasts derived from normal human osteoclast precursor cells were inhibited by a monoclonal antibody specifically binding to Siglec-15 protein.

b) Effect of addition of rat anti-mouse Siglec-15 monoclonal antibody (#32A1) on cell fusion of osteoclasts from normal human osteoclast precursor cells (TRAP staining)

Normal human osteoclast precursor cells (normal human natural osteoclast precursor cells, purchased from Sanko ^Junyaku Co., Ltd., cat. -110) were seeded in 96-well plates. Minimal Essential Medium for Osteoclast Precursor Cells (OPBM, purchased from Sanko Junyaku Co., Ltd., cat. No. PT-8201) as the medium, and the OPGM supplement set contains fetal bovine serum (final concentration 10%), human RANKL (final concentration 68.4ng/ml), human M-CSF (final concentration 33 ng/ml) and the like. To the obtained culture supernatant was added the rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) prepared in Example 6 to give a final concentration of 0.1, 0.3, 1 or 3 μg/ml, at Cells were cultured in a CO ₂ incubator for 3 days. After culturing, the supernatant was removed, and 10% neutral formalin was added to fix the cells. After fixing the cells, wash the cells twice with distilled water, add TRAP staining solution (0.27mM naphthol AS-MX phosphate (manufactured by SigmaCo., Ltd.), 1.6mM fast red purple LB salt (Sigma Co. , Ltd.), 1% dimethylformamide, 50 mM sodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)), and reacted at room temperature for 5 minutes. After the reaction, the cells were washed twice with distilled water, and then observed by microscopy ( FIG. 11 ). As a result, #32A1 antibody inhibited the formation of TRAP-positive multinucleated osteoclasts in a concentration-dependent manner within the range of 0.3 μg/ml to 3 μg/ml.

c) Effect of addition of rat anti-mouse Siglec-15 monoclonal antibody (#32A1) on bone resorption activity of normal human osteoclast precursor cells (evaluation using collagen-coated plates)

Osteoclasts are known to release proteases such as cathepsin K and degrade type I collagen, which is a constituent component of bone tissue. OsteoLyse assay kit (manufactured by Lonza, Inc., catalog number PA-1500) provides a 96-well plate (96-well OsteoLyse cell culture plate) coated with europium-conjugated human The amount of fluorescent collagen fragments released into the supernatant by osteoclasts makes it possible to evaluate the bone resorption activity of osteoclasts in vitro. Normal human osteoclast precursor cells (normal human natural osteoclast precursor cells, purchased from Sanko ^Junyaku Co., Ltd., cat. no. 2T-110) were seeded in 96-well OsteoLyse cell culture plates. Minimal Essential Medium for Osteoclast Precursor Cells (OPBM, purchased from Sanko Junyaku Co., Ltd., cat. No. PT-8201) as the medium, and the OPGM supplement set contains fetal bovine serum (final concentration 10%), human RANKL (final concentration 68.4ng/ml), human M-CSF (final concentration 33 ng/ml) and the like. To the obtained culture supernatant was added the rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) prepared in Example 6 to give a final concentration of 0.1, 0.3, 1 or 3 μg/ml, at Cells were cultured in a CO ₂ incubator for 3 days. A 10 μl aliquot of the culture supernatant was collected, 200 μl of the fluorophore-releasing reagent contained in the OsteoLyse assay kit was added thereto, and the fluorescence intensity (excitation : 340 nm, emission: 615 nm), thereby determining the amount of free fluorescent collagen fragments released in the culture supernatant (Figure 12). As a result, the #32A1 antibody reduced the amount of fluorescent collagen fragments increased by the addition of RANKL in a concentration-dependent manner within the range of 0.3 μg/ml to 3 μg/ml. The results indicated that the bone resorption activity of human osteoclasts was inhibited by the investigated monoclonal antibody specifically binding to Siglec-15 protein.

Example 15

Evaluation of Biological Activity of Rat Anti-Mouse Siglec-15 Monoclonal Antibody Using Ovariectomized Rats

a) Animal experiment methods

Both ovaries were removed from 12-week-old female F344 rats (obtained from Charles River Laboratories Japan, Inc.), and the rats were divided into 3 groups: vehicle administration group; rat anti-mouse Siglec-15 monoclonal antibody# 8A1 administration group; and rat anti-mouse Siglec-15 monoclonal antibody #32A1 administration group. In addition, one group was made into a sham group. In the antibody administration group, the rat anti-mouse Siglec-15 monoclonal antibody #8A1 prepared in Example 6 or the rat anti-mouse Siglec-15 monoclonal antibody #32A1, 3 times a week, repeated for 4 weeks. In the vehicle administration group and the sham operation group, PBS containing 0.01% Tween 20 was intraperitoneally administered as a vehicle. Four weeks after the start of administration, urine was collected under fasting conditions for 24 h, and urine samples were stored at -80 ºC until measurement. After the urine collection was complete, the rats were euthanized and the lumbar spine was dissected from each rat.

b) Measurement of lumbar bone mineral density

The soft tissues connected to the dissected lumbar vertebrae were removed, and the 4th to 6th lumbar vertebrae were extracted. The extracted lumbar vertebra was defatted and dehydrated by shaking in ethanol, then air-dried, and bone mineral density was measured using a bone densitometer (DCS-600EX, manufactured by Aloka Co., Ltd.). The result is shown in Fig. 13(A). A significant decrease in bone mineral density of the lumbar spine was observed in the ovariectomy group compared to the sham group, however, in the #8A1 and #32A1 antibody administration groups, the increase in bone mineral density caused by ovariectomy was significantly suppressed reduce.

c) Measurement of urinary deoxypyridinoline excretion

Metabolites of multiple type I collagen cross-links prominently reflect bone turnover, especially bone resorption. Most importantly, deoxypyridinolines are mainly localized in bone collagen and, therefore, are considered to be very reliable indicators of bone resorption.

A frozen preserved urine sample was thawed, and insoluble matter was precipitated by centrifugation, thereby obtaining a supernatant. The amount of deoxypyridinoline contained in the supernatant was measured using Osteolinks "DPD" (manufactured by DS Pharma Biomedical Co., Ltd.). In addition, by using Creatinine Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), the creatinine content in the supernatant was also measured, and the corrected amount of deoxypyridinoline to creatinine was calculated. The result is shown in Fig. 13(B). Urinary deoxypyridinoline excretion was significantly increased in the ovariectomized group compared to the sham group, thus, indicating increased osteoclastic bone resorption in ovariectomized rats. On the other hand, in the #8A1 and #32A1 antibody-administered groups, the increase in deoxypyridinoline excretion due to ovariectomy was suppressed so that the level of deoxypyridinoline excretion was comparable to that of the sham operation group. From this result, it was also confirmed in animal models that the studied monoclonal antibody that specifically binds to Siglec-15 inhibits osteoclastic bone resorption, and strongly indicates that the inhibition of ovariectomized bone loss is due to the inhibition of bone resorption. Reduction of lumbar bone mineral density in rats.

Example 16

Determination of the binding site (epitope) of the rat anti-mouse Siglec-15 monoclonal antibody

a) Expression and purification of human Siglec-15 V-group domain

Into the vector pDEST14 (Invitrogen, Inc., Cat. No. 11801-016), the DNA encoding the protein in which the His-tag and the thrombin recognition sequence were linked to the human Siglec-15 V-group domain (a A polypeptide comprising the amino acid sequence in the NCBI protein database with accession number NP_998767 or the N-terminal side of amino acid residues 39-165) of the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing. Using this plasmid, Escherichia coli Rosetta-gamiB (DE3) (Novagen, Inc., cat. No. 71136-4) was transformed, and cultured in TB medium (Invitrogen, Inc., cat. No. 22711-022). After culturing, the bacterial cells were homogenized by sonication, the resulting homogenate was centrifuged, and the supernatant was purified using a HisTrap HP column (GE Healthcare, Co., Ltd., catalog number 17-5247-01). Thereafter, the His tag was cleaved with thrombin, and then Mono S5/50 GL column (GE Healthcare, Co., Ltd., catalog number 17-5168-01) and Superdex 75 10/300 column (GE Healthcare, Co., Ltd. ., catalog number 17-5174-01), the human Siglec-15 V-group domain was purified until a single band with a molecular weight of 14 kDa was obtained by electrophoresis.

b) Purification of soluble human Siglec-15-Fc

Soluble human Siglec-15-Fc was purified by the method described in Example 12.

c) Competition ELISA of human V-group domain and rat anti-mouse Siglec-15 monoclonal antibody #32A1

In a 96-well maxi-sorp plate (manufactured by Nunc, Inc., model: 442404), 100 μl of goat anti-human Fc antibody (Jackson ImmunoResearch, Inc., model: 109-005-098) (1.25 μg/ml ) were added to each well and immobilized overnight at 4ºC. After washing the 96-well maxi-sorp plate twice with PBS, 100 μl of soluble human Siglec-15-Fc (1 μg/ml) was added to each well and immobilized at room temperature for 1 hour. Thereafter, 300 μl of 5% skim milk/PBS solution was added to each well, and the wells were blocked for 3 hours at room temperature. At the same time, 2 μg/ml of rat anti-mouse Siglec-15 monoclonal antibody #32A1 was mixed with an equal amount of 0, 0.032, 0.16, 0.8, 4 or 20 μg/ml of human Siglec-15 V-group The domains were mixed and the reaction was allowed to proceed for 1.5 hours at room temperature. After the 96-well maxi-sorp plate had been washed twice with PBS, 100 μl of a solution of mixed rat anti-mouse Siglec-15 monoclonal antibody #32A1 and human Siglec-15 V-group domain was added thereto, And the reaction was allowed to proceed at room temperature for 1 hour. After the 96-well maxi-sorp plate had been washed 5 times with 0.05% Tween 20 (Bio-Rad Laboratories, Inc., Cat. No. 170-6531)/PBS solution (hereinafter referred to as "PBST solution"), added 100 μl of HRP-labeled goat anti-rat IgG antibody (Beckman Coulter, Inc., Cat. No. 732664, diluted to 2000-fold), and the reaction was allowed to proceed at room temperature for 1 hour. After the plate had been washed 5 times with the PBST solution, 100 µl of the developing solution of ELISA POD Substrate ABTS Kit (Nacalai Tesque Co., Ltd., cat. No. 14351-80) was added thereto, and the reaction was allowed to proceed for 30 minutes. Then, 100 µl of a reaction stop solution was added thereto, and the absorbance at 405 nm was measured. The results of the competition ELISA showed that the binding of rat anti-mouse Siglec-15 monoclonal antibody #32A1 to immobilized human Siglec-15 was inhibited in a concentration-dependent manner by the human V-group domain. Thus, it was shown that the epitope of the rat anti-mouse Siglec-15 mAb #32A1 is the human Siglec-15 V-group domain (a domain comprising amino acids in the NCBI protein database with accession number NP_998767 sequence or by amino acid residues 39-165 of the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing) (Figure 14).

Example 17

Amplification of cDNA encoding the variable region of rat anti-mouse Siglec-15 monoclonal antibody #32A1 and its base sequence analysis

a) Preparation of mRNA

According to a) of Example 6, hybridoma #32A1 was cultured. About 65 μg of mRNA was prepared from 4 x 10 ⁷ hybridoma #32A1 cells using QuickPrep mRNA Purification Kit (GEHealthcare, Co., Ltd.).

b) Synthesis of cDNA (5'-RACE-Ready cDNA)

cDNA (5'-RACE-ReadycDNA) was synthesized using PrimeScript Reverse Transcriptase (TaKaRa Bio, Inc.) and SMART RACE cDNA Amplification Kit (Clontech Co., Ltd.) using 0.3 μg of the mRNA prepared in a) .

c) Amplify the cDNA of the heavy chain variable region of the #32A1 gene by 5'-RACE PCR

UPM (Universal Primer A Mix, attached to the SMART RACE cDNA Amplification Kit) and an oligonucleotide with the sequence 5'-GGCCGGGTGGGCTACGTTGCAGGTGACGGTCTG-3' (RG2AR2: SEQ ID NO: 23 in the Sequence Listing) were used as amplified by PCR. Primers for the cDNA of the variable region of the heavy chain gene of #32A1 were added. Using UPM attached to SMART RACE cDNA Amplification Kit (Clontech Co., Ltd.) as UPM, RG2AR2 was designed from the sequence of the rat heavy chain (IgG2a) constant region in the database, and synthesized according to a conventional method.

Using this primer combination and the cDNA (5'-RACE-Ready cDNA) synthesized in b) as a template, the cDNA of the variable region of the heavy chain gene of #32A1 was amplified by 5'-RACE PCR. PCR was performed using the Advantage 2 PCR Kit (Clontech Co., Ltd.), and the conditions of the thermal cycler were set as follows: After heating at 94°C for 1 minute, the temperature cycle of "0.5 minutes at 94°C and 3 minutes at 72°C" was repeated 5 times, then repeat the temperature cycle of "94°C for 0.5 minutes, 70°C for 0.5 minutes and 72°C for 3 minutes" 5 times, and then repeat the temperature cycle of "94°C for 0.5 minutes, 68°C for 0.5 minutes and 72°C for 3 minutes" 20 times , and then incubated at 4 °C.

d) Amplify #32A1 light chain gene variable region cDNA by 5'-RACE PCR

UPM (Universal Primer A Mixture, attached to the SMART RACE cDNA Amplification Kit) and an oligonucleotide having the sequence 5'-CATGCTGTACGTGCTGTCTTTGCTGTCCTGATCAG-3' (RKR2: SEQ ID NO: 24 in the Sequence Listing) were used as amplified by PCR. Primers for the cDNA of the variable region of the #32A1 light chain gene. Using UPM attached to SMART RACE cDNA Amplification Kit (Clontech Co., Ltd.) as UPM, RKR2 was designed from the sequence of the rat light chain (κ chain) constant region in the database, and synthesized according to a conventional method.

Using this primer combination and the cDNA (5'-RACE-Ready cDNA) synthesized in b) as a template, the cDNA of the variable region of the light chain gene of #32A1 was amplified by 5'-RACE PCR. PCR was performed using the Advantage 2 PCR Kit (Clontech Co., Ltd.), and the conditions of the thermal cycler were set as follows: After heating at 94°C for 1 minute, the temperature of "94°C for 0.5 minutes and 72°C for 3 minutes" was repeated Cycle 5 times, then repeat the temperature cycle of "94°C for 0.5 minutes, 70°C for 0.5 minutes and 72°C for 3 minutes" 5 times, and repeat the temperature cycle of "94°C for 0.5 minutes, 68°C for 0.5 minutes and 72°C for 3 minutes" for 20 times, and then incubated at 4°C.

e) Determination of the base sequences of the cDNAs of the heavy chain and light chain variable regions

The cDNA of the heavy chain variable region amplified in c) was purified using MinElute PCR Purification Kit (QIAGEN Inc.), followed by sequence analysis of the DNA sequence. From the sequence design of the rat heavy chain (IgG2a) constant region in the database, the oligonucleotide with the sequence 5'-CTCCAGAGTTCCAGGTCACGGTGACTGGC-3' (RG2AR3: SEQ ID NO: 25 in the sequence listing) was used as a sequencing primer, and according to the routine method synthesis.

The cDNA of the light chain variable region amplified in d) was purified using MinElute PCR Purification Kit (QIAGEN Inc.), followed by sequence analysis of the DNA sequence. From the sequence of the constant region of the rat light chain (κ chain) in the database, an oligonucleotide with the sequence 5'-TCCAGTTGCTAACTGTTCCG-3' (sqRK: SEQ ID NO: 26 in the sequence listing) was designed as a sequencing primer, and Synthesized according to conventional methods.

The cDNA containing the heavy chain variable region obtained by sequence analysis has a base sequence represented by SEQ ID NO: 27 in the Sequence Listing and encodes an amino acid sequence represented by SEQ ID NO: 28 in the Sequence Listing. The amino acid sequence represented by amino acid numbers 1-19 of SEQ ID NO: 28 corresponds to the secretion signal, the amino acid sequence represented by its amino acid numbers 20-140 corresponds to the heavy chain variable region, and the amino acid sequence represented by its amino acid numbers 141-167 Correspondence focuses on the constant region (part) of the heavy chain. The above heavy chain variable region comprises: CDRH1 (DYFMN) comprising the amino acid sequence represented by SEQ ID NO: 44, CDRH2 (QIRNKIYTYATFYAESLEG) comprising the amino acid sequence represented by SEQ ID NO: 45, and comprising the amino acid sequence represented by SEQ ID NO: 46 represents the amino acid sequence of CDRH3 (SLTGGDYFDY). In addition, the cDNA containing the light chain variable region has a base sequence represented by SEQ ID NO: 29 in the Sequence Listing and encodes an amino acid sequence represented by SEQ ID NO: 30 in the Sequence Listing. The amino acid sequence represented by amino acid numbers 1-20 of SEQ ID NO: 30 corresponds to the secretion signal, the amino acid sequence represented by its amino acid numbers 21-132 corresponds to the light chain variable region, and the amino acids represented by its amino acid numbers 133-139 The sequence corresponds to the light chain constant region (part). The above light chain variable region comprises: CDRL1 (RASQSVTISGYSFIH) comprising the amino acid sequence represented by SEQ ID NO: 47, CDRL2 (RASNLAS) comprising the amino acid sequence represented by SEQ ID NO: 48, and comprising the amino acid sequence represented by SEQ ID NO: 49 The amino acid sequence of CDRL3(QQSRKSPWT) is indicated.

Example 18

Preparation of Human Chimeric Antibody Gene Expression Constructs of Rat Anti-Mouse Siglec-15 Monoclonal Antibody #32A1

a) Preparation of general expression vectors pEF1/FCCU-1 and pEF6KCL

a)-i) Construction of human light chain expression vector pEF6KCL

Using plasmid pEF6/V5-HisB (Invitrogen) as a template, and using the following primers to perform PCR, a DNA fragment immediately downstream of BGHpA (2174) to SmaI (2958) (DNA containing the f1 origin of replication and the SV40 promoter and origin Fragment, hereinafter referred to as "Fragment A"):

5'-CCACGCGCCCTGTAGCGGCGCATTAAGC-3' (Primer EFF1: SEQ ID NO:31 in the Sequence Listing)

5'-AAACCCGGGAGCTTTTTGCAAAAGCCTAGG-3' (Primer EFsmaR: SEQ ID NO: 32).

The resulting fragment A and a DNA fragment (SEQ ID NO: 33, hereinafter referred to as "fragment A") containing the DNA sequence encoding the human κ chain secretion signal, the human κ chain constant region, and the human polyadenylic acid extra signal were ligated by overlapping PCR. B"). The thus obtained DNA fragment in which fragment A and fragment B were ligated (hereinafter referred to as "fragment A+B") was digested with restriction enzymes KpnI and SmaI, and ligated into plasmid pEF6/V5 digested with restriction enzymes KpnI and SmaI -HisB (Invitrogen), from which a human light chain expression vector pEF6KCL was constructed with a signal sequence, a cloning site, a human kappa chain constant region, and a human polyadenylation additional signal sequence downstream of the EF1 promoter.

a)-ii) Construction of pEF1/KCL

Plasmid pEF1/KCL was constructed by cleaving pEF6KCL obtained by the above method with restriction enzymes KpnI and SmaI to obtain a DNA fragment, which was ligated to pEF1/myc-HisB (Invitrogen, Inc.) digested with KpnI and SmaI.

a)-iii) Construction of human heavy chain expression vector pEF1/FCCU-1

A DNA fragment containing the DNA sequence encoding the human IgG1 signal sequence and constant region amino acid sequence (SEQ ID NO: 34 in the Sequence Listing) was digested with restriction enzymes NheI and PmeI, and ligated into plasmid pEF1/KCL digested with NheI and PmeI , thereby constructing a human heavy chain expression vector pEF1/FCCU-1, which has a signal sequence, a cloning site, a human heavy chain constant region, and a human polyadenylation additional signal sequence downstream of the EF1 promoter.

b) Preparation of #32A1 Human Chimeric Antibody Heavy Chain Expression Construct

According to a conventional method, an oligonucleotide having the sequence 5'-aaagctgagcGAGGTGCAAATTT TGGAGACTGGAGGAGGC-3' (32A1HF: SEQ ID NO: 35 in the sequence listing) and an oligonucleotide having the sequence 5'-aaagctgagctGACTGTGACCATGACTC CTTGGCCCCAG-3' (32A1HR: in the sequence listing) were synthesized. The oligonucleotide of SEQ ID NO: 36) was used as a primer for amplifying the cDNA of #32A1 heavy chain variable region by PCR.

Furthermore, in order to integrate the PCR product into pEF1/FCCU-1, these primers were digested, thereby adding the recognition sequence for the restriction enzyme BlpI. Using this primer combination and the cDNA of the heavy chain variable region purified in e) of Example 17 as a template, PCR was performed according to a conventional method. Using the MinElute PCR Purification Kit (QIAGEN, Inc.), the obtained PCR product was purified, and then digested with the restriction enzyme BlpI (NEW ENGLAND BIOLABS, Inc.) to obtain a DNA fragment, which was digested with the restriction enzyme BlpI The site was inserted into pEF1/FCCU-1, thereby constructing the #32A1 human chimeric antibody heavy chain expression construct. The sequence of the insert was confirmed by sequence analysis. According to a conventional method, an oligonucleotide having the sequence 5'-TAATACGACTCACTATAGGG-3' (F11: SEQ ID NO: 37 in the sequence listing) was synthesized as a primer for sequencing. The expression vector into which the insert was correctly inserted was named "32A1H/pEF1/FCCU".

c) Preparation of #32A1 Human Chimeric Antibody Light Chain Expression Construct

According to a conventional method, an oligonucleotide having the sequence 5'-aaacatggcGACATTGTCTTG ACCCAGTCTCCTGCTTTGG-3' (32A1LF: SEQ ID NO: 38 in the sequence listing) and an oligonucleotide having the sequence 5'-aaacgtacgTCTCAATTCCAGCTTGGTGCCTCCAGCG-3' (32A1LR: in the sequence listing) were synthesized. The oligonucleotide of SEQ ID NO: 39) was used as a primer for PCR amplification of the cDNA of #32A1 light chain variable region.

Furthermore, in order to integrate the PCR product into pEF6KCL, these primers were digested, thereby adding the recognition sequence of the restriction enzyme NdeI to 32A1LF and the recognition sequence of the restriction enzyme BsiWI to 32A1LR. Using this primer combination and the cDNA of the light chain variable region purified in e) of Example 17 as a template, PCR was performed according to a conventional method. Using the MinElute PCR Purification Kit (QIAGEN, Inc.), the resulting PCR product was purified, and then a DNA fragment was obtained by digesting the PCR product with restriction enzymes NdeI (TaKaRa Bio, Inc.) and BsiWI (NEW ENGLAND BIOLABS, Inc.), This was inserted into pEF6KCL at the site cleaved with restriction enzymes NdeI (TaKaRa Bio, Inc.) and BsiWI (NEW ENGLAND BIOLABS, Inc.), thereby constructing a #32A1 human chimeric antibody light chain expression construct. The sequence of the insert was confirmed by sequence analysis. Primer F11 represented by SEQ ID NO: 37 in the Sequence Listing was used as a primer for sequencing. The expression vector into which the insert fragment was correctly inserted was named "32A1L/pEF6KCL".

The #32A1 human chimeric antibody heavy chain gene prepared in b) has a nucleotide sequence represented by SEQ ID NO: 40 in the Sequence Listing, and encodes an amino acid sequence represented by SEQ ID NO: 41 in the Sequence Listing. The amino acid sequence represented by amino acid numbers 1-19 of SEQ ID NO: 41 corresponds to the secretion signal, the amino acid sequence represented by its amino acid numbers 20-140 corresponds to the heavy chain variable region, and the amino acid sequence represented by its amino acid numbers 141-470 Corresponding to corresponding to the constant region of the heavy chain. In addition, the #32A1 human chimeric antibody light chain gene prepared in c) has a nucleotide sequence represented by SEQ ID NO: 42 in the Sequence Listing, and encodes an amino acid represented by SEQ ID NO: 43 in the Sequence Listing sequence. The amino acid sequence represented by amino acid numbers 1-20 of SEQ ID NO: 43 corresponds to the secretion signal, the amino acid sequence represented by its amino acid numbers 21-132 corresponds to the light chain variable region, and the amino acids represented by its amino acid numbers 133-237 The sequence corresponds to the light chain constant region.

Example 19

Preparation of human chimeric antibody against rat anti-mouse Siglec-15 monoclonal antibody #32A1

a) Preparation of human chimeric antibody

Inoculate 3 x 10 ⁷ cells of 293 FreeStyle cells in logarithmic growth phase into 30 ml of fresh FreeStyle 293 Expression Medium (Invitrogen, Inc.) (4 batches were prepared, with 30-ml culture as 1 batch) , and cultured with shaking (125 rpm) at 37ºC in an 8% CO ₂ incubator. 300 μg of polyethyleneimine (manufactured by Polyscience, Inc., #24765) was dissolved in 1 ml of Opti-Pro SFM medium (manufactured by Invitrogen, Inc.), and the resulting solution was left at room temperature for 5 minutes. Using the PureLink HiPure Plasmid Kit (Invitrogen, Inc.), prepare the #32A1 human chimeric antibody heavy chain expression vector 32A1H/pEF1/FCCU and #32A1 human chimeric antibody light chain expression vector 32A1L/pEF6KCL produced in Example 18 . 32A1H/pEF1/FCCU (15 μg) and 32A1L/pEF6KCL (45 μg) were suspended in 1 ml Opti-Pro SFM medium (Invitrogen, Inc.), and the resulting suspension was added to 1 ml polyethyleneimine/Opti -Pro SFM mixture (already at room temperature for 5 minutes), the resulting mixture was left at room temperature for another 5 minutes. Then, add 2 ml of polyethyleneimine/expression vector/Opti-Pro SFM mixture into each batch of 293 FreeStyle cell suspension, and continue shaking culture. After culturing the cells for 7 days at 37 ºC in 8% _CO , collect the culture supernatant from each batch.

b) Purification of Human Chimeric Antibody

90 ml of the above-obtained culture supernatant (3 lots) was filtered through a filter (manufactured by NALGENE, Inc., #295-4545), and 0.5 ml of MabSelect SuRe (GE Healthcare Bio-science Co., Ltd., #17-5438-01) was added to the filtrate, and the resulting mixture was shaken at 80 rpm and 10ºC overnight. The next day, the carrier was collected and washed with PBS, after which the antibody was eluted with 1 M arginine solution (pH 4.0). The eluate was loaded onto a PD-10 column (manufactured by GE Healthcare Bio-science Co., Ltd., #17-0851-01), the liquid was replaced with PBS, and then Amicon Ultra-4 (manufactured by Millipore Co., Ltd. , #UFC805008) was concentrated, thereby obtaining 1.2 ml of human chimeric antibody (0.98 mg/ml). The concentration of the antibody was calculated from the measurement at 280 nm using a Hitachi Diode Array BioPhotometer U-0080D.

Example 20

Evaluation of the Binding Properties of Human Chimeric Antibody of Rat Anti-Mouse Siglec-15 Monoclonal Antibody #32A1 to Mouse Siglec-15 Protein

Competitive inhibition of the purified human chimeric antibody prepared in Example 19 by the rat #32A1 antibody was determined by the following method. Using PBS, the mouse Siglec-15-Fc purified in Example 4 was diluted to 1 μg/ml, and the diluted solution was distributed on an immunoplate (manufactured by Nunc, Inc., #437111) at 100 μl/well , and leave the plate overnight at 4ºC to allow the protein to adsorb to the plate. The next day, each well was washed twice with PBS-T solution (PBS, 0.05% (v/v) Tween 20), at 350 μl/well, distributed by diluting skim milk (Morinaga Milk Industry Co., Ltd. production) to 5% of the resulting solution, and the plate was left to stand at room temperature for 2 hours. The liquid in each well was removed, and each well was washed 3 times with PBS-T solution. Thereafter, a mixed solution (PBS solution containing skim milk at a final concentration of 0.5%) containing 0.25 μg/ml of human chimeric antibody and different concentrations (0 μg/ml, 0.016 μg/ml, 0.05 μg/ml, 0.16 μg/ml, 0.5 μg/ml, 1.66 μg/ml or 5 μg/ml) #32A1 antibody or rat control IgG antibody (manufactured by R&D Systems, Inc., #6- 001-A), and the plate was left to stand at room temperature for 2 hours. After washing each well 3 times with PBS-T solution, add alkaline phosphatase- Conjugated AffiniPure goat anti-human IgG (manufactured by Jackson ImmunoResearch, Inc., #109-055-097), and the plate was left to stand at room temperature for 1 hour. The liquid in each well was removed, and each well was washed 5 times with PBS-T solution. Thereafter, a fluorescent substrate solution (manufactured by Roche Co., Ltd., #11681982001) was added at 100 μl/well, and a fluorescent reaction was performed. Ten minutes after the addition of the fluorescent substrate solution, the fluorescence intensity was measured using a plate reader. The results showed that #32A1 antibody inhibited the binding of human chimeric antibody to mouse Siglec-15-Fc in a concentration-dependent manner. In addition, about 40% competitive inhibition was exhibited when the #32A1 antibody and the human chimeric antibody were mixed at the same concentration (1:1). Therefore, the #32A1 antibody and the human chimeric antibody were considered to have substantially the same affinity for mouse Siglec-15-Fc. On the other hand, rat control IgG showed no competitive inhibition (Figure 15).

Example 21

Biological activity of human chimeric antibody against rat anti-mouse Siglec-15 monoclonal antibody #32A1 evaluated based on mouse osteoclastogenesis assay and human osteoclastogenesis assay

a) Mouse osteoclast formation assay

Using the human chimeric antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 19, the effect on osteoclast differentiation of mouse bone marrow non-adherent cells was examined. In α-MEM medium containing 10% FBS and 10 ng/ml M-CSF (manufactured by R&D Systems, Inc.), at ^1.5 x 105 cells/ml, the mouse prepared by the method of Example 8 was prepared Bone marrow non-adherent cells, the resulting cell preparation was inoculated into each well of a 96-well plate in an amount of 200 μl, and the cells were cultured in a CO ₂ incubator for 2 days. The old culture solution in the 96-well plate was removed, and 100 μl of MEM-α medium containing 10% FBS to which human RANKL (RANKL, Peprotech, Inc.) and M-CSF at a final concentration of 20 ng/ml and 10 ng/ml, respectively. To this cell culture medium, the rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 6 or the rat anti- -Human chimeric antibody to mouse Siglec-15 monoclonal antibody #32A1, cells were cultured for an additional 3 days in a CO ₂ incubator. After the end of the culture, the tartrate-resistant acid phosphatase (TRAP) activity of the formed osteoclasts was measured by the method described in Example 9. After the enzymatic reaction was stopped, the absorbance at 405 nm of each well was measured, and the resulting measurement was used as an index of TRAP activity. The result is shown in Figure 16. In the case of the #32A1 antibody and the human chimeric antibody of the rat anti-mouse Siglec-15 monoclonal antibody #32A1, a dose-dependent inhibition of TRAP activity was observed at a concentration of 50 ng/ml or higher. This result shows that the human chimeric antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1 has an activity of inhibiting osteoclast formation (differentiation and maturation of osteoclasts) substantially equivalent to that of the rat #32A1 antibody.

b) Evaluation using normal human osteoclast precursor cells (TRAP staining)

Normal human osteoclast precursor cells (normal human natural osteoclast precursor cells, purchased from Sanko ^Junyaku Co., Ltd., cat. no. 2T-110) were seeded in 96-well plates. Minimal Essential Medium for Osteoclast Precursor Cells (OPBM, purchased from Sanko Junyaku Co., Ltd., cat. No. PT-8201) as the medium, and the OPGM supplement set contains fetal bovine serum (final concentration 10%), human RANKL (final concentration 66 ng/ml), human M-CSF (final concentration 33 ng/ml) and the like. The human chimeric antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 19 was added to the obtained culture supernatant at a final concentration of 0.3, 1, 3 or 10 μg/ml, Cells were cultured for 3 days in a _CO incubator. After culturing, the supernatant was removed, and 10% neutral formalin was added to fix the cells. After fixing the cells, wash the cells twice with distilled water, add TRAP staining solution (0.27mM naphthol AS-MX phosphate (manufactured by SigmaCo., Ltd.), 1.6mM fast red purple LB salt (Sigma Co. , Ltd.), 1% dimethylformamide, 50 mM sodium tartrate, 0.1 M sodium acetate buffer (pH 5.0)), and reacted at room temperature for 5 minutes. After the reaction, the cells were washed twice with distilled water, and then observed by microscopy ( FIG. 17 ). As a result, the formation of TRAP-positive multinucleated osteoclasts was inhibited by adding the human chimeric antibody of #32A1 in the range of 0.3 μg/ml to 10 μg/ml.

Example 22

Humanized Antibody Design of Rat Anti-Mouse Siglec-15 Monoclonal Antibody #32A1 (1)

a) Design of humanized form of #32A1

a)-i) Molecular modeling of the variable region of #32A1

Molecular modeling of the variable region of #32A1 was performed by a method commonly referred to as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). The basic sequence of the human immunoglobulin variable region registered in the protein database (Nuc. Acid Res. 35, D301-D303 (2007)) (the three-dimensional structure derived from the X-ray crystal structure can be obtained) and the above determination Relative comparison of the variable region of #32A1. As a result, among antibodies with similar deletions in framework, 1LK3 was chosen as having the highest sequence homology to the light chain variable region of #32A1. Furthermore, 1AD0 was chosen as having the highest sequence homology to the heavy chain variable region of #32A1. A "framework model" was obtained by combining the coordinates of 1LK3 and 1AD0 corresponding to the light and heavy chains of #32A1, generating the three-dimensional structure of the framework regions. According to the taxonomy of Thornton et al. (J. Mol. Biol., 263, 800-815, (1996)), the CDRs of class #32A1 are as follows: CDRL1, CDRL2, CDRL3, CDRH1 and CDRH2 are assigned to clusters 15A, 7A, respectively , 9A, 10A and 12B. According to the H3-rule (FEBSletter 399, 1-8 (1996)), CDRH3 is classified as k(8)C. Then, representative conformations of each CDR were integrated into the framework model.

Finally, to obtain a possible molecular model of the variable region of #32A1 in terms of energy, energy calculations were performed to exclude unfavorable interatomic contacts. The above operations were performed using the commercially available three-dimensional protein structure prediction program Prime and the equivalent search program MacroModel (Schrodinger, LLC).

a)-ii) Design of the amino acid sequence of humanized #32A1

Humanized #32A1 antibody was constructed according to a method generally called CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Recipient antibodies were selected in two ways based on amino acid homology within the framework regions. The framework region sequence of #32A1 was compared with the sequence of all human frameworks in the Kabat database (Nuc. Acid Res. 29, 205-206 (2001)) containing antibody amino acid sequences. As a result, the M37GO37'CL antibody was selected as the acceptor based on the 73% sequence homology in the framework region. Amino acid residues in the framework region of M37GO37'CL were aligned with those of #32A1, identifying positions where different amino acids were used. Using the 3D model of #32A1 constructed above, the positions of these residues were analyzed. The donor residues to be grafted onto the recipient are then selected according to the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)).

The framework region sequence of #32A1 was compared with all human framework sequences in IgBLAST (Nuc. Acid Res.36, D25-D30 (2007)). As a result, for the L-chain, AAB34430 was selected as the acceptor based on the 79% sequence homology in the framework region. For the H-chain, CAF31288 was chosen as the acceptor based on the 78% sequence homology in the framework regions. Amino acid residues in the framework region of AAB34430 (for the L-chain) and CAF31288 for the framework region (for the H-chain) were compared with those of #32A1, identifying where different amino acids were used Location. Using the 3D model of #32A1 constructed above, the positions of these residues were analyzed. The donor residues to be grafted onto the recipient are then selected according to the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). In these methods, the humanized #32A1 sequence was constructed as described in the Examples below by transferring some selected donor residues to the acceptor antibody.

b) Humanization of #32A1 heavy chain

b)-i) h#32A1-T1H-type heavy chain:

By using leucine, valine, serine, glutamine, alanine, glycine, phenylalanine, asparagine, alanine, lysine, asparagine, leucine, methylthio Amino acid number 23 (isoleucine) of the #32A1 heavy chain represented by SEQ ID NO: 28 in the Sequence Listing is replaced by amino acid, asparagine, valine, alanine, threonine, leucine and serine ), 24 (leucine), 26 (threonine), 32 (lysine), 43 (threonine), 61 (glutamic acid), 89 (valine), 95 (aspartic acid ), 96 (serine), 97 (glutamic acid), 98 (serine), 100 (valine), 104 (valine), 105 (serine), 114 (isoleucine), 118 (threonine Acid), 134 (valine), 135 (methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T1H-type heavy chain".

The amino acid sequence of the h#32A1-T1 H-type heavy chain is represented by SEQ ID NO: 51 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 51, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-470 correspond to the signal sequence, heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 51 is represented by SEQ ID NO: 50 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 50, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1410 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 50 and the amino acid sequence of SEQ ID NO: 51 are also shown in FIG. 27 .

b)-ii) h#32A1-T2H-type heavy chain:

By using leucine, valine, serine, glutamine, alanine, asparagine, alanine, lysine, asparagine, leucine, methionine, valine, Alanine, threonine, leucine and serine replace amino acid numbers 23 (isoleucine), 24 (leucine), 26 ( threonine), 32 (lysine), 43 (threonine), 95 (aspartic acid), 96 (serine), 97 (glutamic acid), 98 (serine), 100 (valine) , 104 (valine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 (methionine) and 140 (leucine) designed humanized The #32A1 heavy chain was named "h#32A1-T2H-type heavy chain".

The amino acid sequence of the h#32A1-T2H-type heavy chain is represented by SEQ ID NO: 53 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 53, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-470 correspond to the signal sequence, the heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 53 is represented by SEQ ID NO: 52 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 52, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1410 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 52 and the amino acid sequence of SEQ ID NO: 53 are also shown in FIG. 28 .

b)-iii) h#32A1-T3H-type heavy chain:

By replacing the amino acids of #32A1 heavy chain represented by SEQ ID NO: 28 in the sequence listing with valine, serine, glutamine, methionine, valine, threonine, leucine and serine, respectively No. 24 (leucine), 26 (threonine), 32 (lysine), 104 (valine), 114 (isoleucine), 134 (valine), 135 (methionine ) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T3H-type heavy chain".

The amino acid sequence of the h#32A1-T3H-type heavy chain is represented by SEQ ID NO: 55 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 55, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-470 correspond to the signal sequence, the heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 55 is represented by SEQ ID NO: 54 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 54, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1410 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 54 and the amino acid sequence of SEQ ID NO: 55 are also shown in FIG. 29 .

b)-iv) h#32A1-T5H-type heavy chain:

By using valine, valine, serine, glutamine, glycine, phenylalanine, asparagine, lysine, threonine, methionine, asparagine, valine, threonine amino acid number 23 (isoleucine), 24 (leucine), 26 (threonine), 32 of #32A1 heavy chain represented by SEQID NO: 28 in the sequence listing, leucine and serine (lysine), 61 (glutamic acid), 89 (valine), 95 (aspartic acid), 97 (glutamic acid), 99 (serine), 104 (valine), 105 (serine ), 114 (isoleucine), 134 (valine), 135 (methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1- T5H-Type Heavy Chain".

The amino acid sequence of the h#32A1-T5H-type heavy chain is represented by SEQ ID NO: 57 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 57, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-470 correspond to the signal sequence, the heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 57 is represented by SEQ ID NO: 56 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 56, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1410 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 56 and the amino acid sequence of SEQ ID NO: 57 are also shown in Figure 30.

b)-v) h#32A1-T6H-type heavy chain:

by substituting valine, valine, serine, glutamine, asparagine, lysine, threonine, methionine, valine, threonine, leucine, and serine by the sequence The amino acid number 23 (isoleucine), 24 (leucine), 26 (threonine), 32 (lysine), 95 (asparagine) of the #32A1 heavy chain represented by SEQ ID NO: 28 in the list amino acid), 97 (glutamic acid), 99 (serine), 104 (valine), 114 (isoleucine), 134 (valine), 135 (methionine) and 140 (leucine Acid) designed humanized #32A1 heavy chain was named "h#32A1-T6H-type heavy chain".

The amino acid sequence of the h#32A1-T6H-type heavy chain is represented by SEQ ID NO: 59 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 59, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-470 correspond to the signal sequence, heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 59 is represented by SEQ ID NO: 58 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 58, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1410 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 58 and the amino acid sequence of SEQ ID NO: 59 are also shown in FIG. 31 .

b)-vi) h#32A1-T7H-type heavy chain:

By separately using leucine, valine, serine, glutamine, alanine, phenylalanine, asparagine, alanine, lysine, asparagine, leucine, methionine , asparagine, valine, alanine, threonine, leucine and serine were replaced by amino acid number 23 (isoleucine) of #32A1 heavy chain represented by SEQ ID NO: 28 in the sequence listing, 24 (leucine), 26 (threonine), 32 (lysine), 43 (threonine), 89 (valine), 95 (aspartic acid), 96 (serine), 97 ( glutamic acid), 98 (serine), 100 (valine), 104 (valine), 105 (serine), 114 (isoleucine), 118 (threonine), 134 (valine) , 135 (methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T7H-type heavy chain".

The amino acid sequence of the h#32A1-T7H-type heavy chain is represented by SEQ ID NO: 72 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 72 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 72 is also shown in Figure 38.

b)-vii) h#32A1-T8H-type heavy chain:

By separately using leucine, valine, serine, glutamine, alanine, glycine, asparagine, alanine, lysine, asparagine, leucine, methionine, asparagine Amide, valine, alanine, threonine, leucine and serine replace amino acid numbers 23 (isoleucine), 24 (leucine) of #32A1 heavy chain represented by SEQ ID NO: 28 in the sequence listing amino acid), 26 (threonine), 32 (lysine), 43 (threonine), 61 (glutamic acid), 95 (aspartic acid), 96 (serine), 97 (glutamic acid ), 98 (serine), 100 (valine), 104 (valine), 105 (serine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 ( Methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T8H-type heavy chain".

The amino acid sequence of the h#32A1-T8H-type heavy chain is represented by SEQ ID NO: 73 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 73 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 73 is also shown in Figure 38.

b-viii) h#32A1-T9H-type heavy chain:

By using leucine, valine, serine, glutamine, alanine, glycine, phenylalanine, asparagine, alanine, lysine, asparagine, leucine, methylthio amino acid number 23 (isoleucine), 24 ( leucine), 26 (threonine), 32 (lysine), 43 (threonine), 61 (glutamic acid), 89 (valine), 95 (aspartic acid), 96 ( serine), 97 (glutamic acid), 98 (serine), 100 (valine), 104 (valine), 114 (isoleucine), 118 (threonine), 134 (valine) , 135 (methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T9H-type heavy chain".

The amino acid sequence of the h#32A1-T9H-type heavy chain is represented by SEQ ID NO: 74 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 74 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 74 is also shown in Figure 38.

b)-ix) h#32A1-T10H-type heavy chain:

By using leucine, valine, serine, glutamine, alanine, asparagine, alanine, lysine, asparagine, leucine, methionine, asparagine, Valine, alanine, threonine, leucine and serine replace amino acid numbers 23 (isoleucine), 24 (leucine) of #32A1 heavy chain represented by SEQ ID NO: 28 in the sequence listing ), 26 (threonine), 32 (lysine), 43 (threonine), 95 (aspartic acid), 96 (serine), 97 (glutamic acid), 98 (serine), 100 ( valine), 104 (valine), 105 (serine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 (methionine) and 140 (leucine amino acid) designed humanized #32A1 heavy chain was named "h#32A1-T10H-type heavy chain".

The amino acid sequence of the h#32A1-T10H-type heavy chain is represented by SEQ ID NO: 75 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 75 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 75 is also shown in Figure 39.

b)-x) h#32A1-T11H-type heavy chain:

By separately using leucine, valine, serine, glutamine, alanine, phenylalanine, asparagine, alanine, lysine, asparagine, leucine, methionine , valine, alanine, threonine, leucine and serine replace the amino acid numbers 23 (isoleucine), 24 (leucine) of #32A1 heavy chain represented by SEQ ID NO: 28 in the sequence listing acid), 26 (threonine), 32 (lysine), 43 (threonine), 89 (valine), 95 (aspartic acid), 96 (serine), 97 (glutamic acid) , 98 (serine), 100 (valine), 104 (valine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 (methionine) and 140 (leucine) designed humanized #32A1 heavy chain was named "h#32A1-T11H-type heavy chain".

The amino acid sequence of the h#32A1-T11 H-type heavy chain is represented by SEQ ID NO: 76 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 76 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 76 is also shown in Figure 39.

b)-xi) h#32A1-T12H-type heavy chain:

By separately using leucine, valine, serine, glutamine, alanine, glycine, asparagine, alanine, lysine, asparagine, leucine, methionine, valine Acid, alanine, threonine, leucine and serine replace amino acid residues 23 (isoleucine), 24 (leucine) of #32A1 heavy chain represented by SEQ ID NO: 28 in the Sequence Listing , 26 (threonine), 32 (lysine), 43 (threonine), 61 (glutamic acid), 95 (aspartic acid), 96 (serine), 97 (glutamic acid), 98 (serine), 100 (valine), 104 (valine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 (methionine) and 140 ( Leucine) designed humanized #32A1 heavy chain was named "h#32A1-T12H-type heavy chain".

The amino acid sequence of the h#32A1-T12H-type heavy chain is represented by SEQ ID NO: 77 in the Sequence Listing. A sequence comprising amino acid residues 1-121 of the amino acid sequence of SEQ ID NO: 77 and a sequence comprising amino acid residues 122-451 thereof correspond to a heavy chain variable region and a heavy chain constant region, respectively. The amino acid sequence of SEQ ID NO: 77 is also shown in Figure 39.

c) Humanization of #32A1 light chain

c)-i) h#32A1-T1L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine , valine, tyrosine, glycine, valine, isoleucine, lysine and threonine are replaced by the amino acid number 24 of the #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing (leucine amino acid), 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 68 (arginine), 81 (isoleucine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine) , 108 (threonine), 110 (phenylalanine), 122 (alanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) The designed humanized #32A1 light chain was named "h#32A1-T1L-type light chain".

The amino acid sequence of the h#32A1-T1 L-type light chain is represented by SEQ ID NO: 61 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 61, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 61 is represented by SEQ ID NO: 60 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 60, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 60 and the amino acid sequence of SEQ ID NO: 61 are also shown in Figure 32.

c)-ii) h#32A1-T2L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, valine, serine, serine, leucine, glutamic acid, valine, valine, valine , isoleucine, lysine and threonine replace amino acid numbers 24 (leucine), 29 (alanine), 29 and 30 of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing Between (i.e. inserting residues deleted in the rat framework), 36 (glutamine), 66 (glutamine), 81 (isoleucine), 99 (asparagine), 100 (proline ), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 127 (leucine), 129 (leucine), 130 (arginine Acid) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T2L-type light chain".

The amino acid sequence of the h#32A1-T2L-type light chain is represented by SEQ ID NO: 63 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 63, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 63 is represented by SEQ ID NO: 62 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 62, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 62 and the amino acid sequence of SEQ ID NO: 63 are also shown in FIG. 33 .

c)-iii) h#32A1-T3L-type light chain:

from the Sequence Listing by substituting aspartic acid, serine, glutamic acid, serine, serine, leucine, glutamic acid, valine, valine, isoleucine, lysine and threonine, respectively Amino acid numbers 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine), 99 of #32A1 light chain represented by SEQ ID NO: 30 (Asparagine), 100 (Proline), 101 (Valine), 104 (Aspartic Acid), 106 (Isoleucine), 127 (Leucine), 129 (Leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T3L-type light chain".

The amino acid sequence of the h#32A1-T3 L-type light chain is represented by SEQ ID NO: 65 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 65, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 65 is represented by SEQ ID NO: 64 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 64, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 64 and the amino acid sequence of SEQ ID NO: 65 are also shown in Figure 34.

c)-iv) h#32A1-T4L-type light chain:

By replacing #32A1 represented by SEQ ID NO: 30 in the sequence listing with glutamic acid, serine, serine, leucine, glutamic acid, valine, isoleucine, lysine and threonine, respectively Amino acid number of light chain 36 (glutamine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chains were named "h#32A1-T4L-type light chain".

The amino acid sequence of the h#32A1-T4 L-type light chain is represented by SEQ ID NO: 67 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 67, the sequence comprising its amino acid residues 21-132 and the sequence comprising its amino acid residues 133-237 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 67 is represented by SEQ ID NO: 66 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 66, the sequence comprising its nucleotides 61-396 and the sequence comprising its nucleotides 397-711 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 67 are also shown in Figure 35.

c)-v) h#32A1-T5L-type light chain:

By using aspartic acid, serine, glutamic acid, asparagine, proline, lysine, serine, serine, leucine, glutamic acid, valine, tyrosine, glycine, glutamine Amide, valine, isoleucine, lysine and threonine replace amino acid numbers 29 (alanine), 29 and 30 of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing (i.e. insertion of residues deleted in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 68 (arginine), 99 (asparagine), 100 (proline amino acid), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 110 (phenylalanine), 122 (alanine), 123 (glycine), 127 (leucine amino acid), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T5L-type light chain".

The amino acid sequence of the h#32A1-T5L-type light chain is represented by SEQ ID NO: 69 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 69, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 69 is represented by SEQ ID NO: 68 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 68, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 68 and the amino acid sequence of SEQ ID NO: 69 are also shown in Figure 36.

c)-vi) h#32A1-T6L-type light chain:

by aspartic acid, serine, glutamic acid, proline, serine, serine, leucine, glutamic acid, valine, valine, isoleucine, lysine and threonine Replacement of amino acid numbers 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing amide), 66 (glutamine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 127 (leucine amino acid), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T6L-type light chain".

The amino acid sequence of the h#32A1-T6L-type light chain is represented by SEQ ID NO: 71 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQ ID NO: 71, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 71 is represented by SEQ ID NO: 70 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 70, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain can Variable region sequences and light chain constant region sequences. The nucleotide sequence of SEQ ID NO: 70 and the amino acid sequence of SEQ ID NO: 71 are also shown in Figure 37.

c)-vii) h#32A1-T7L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine, valine , tyrosine, glycine, valine, isoleucine, lysine and threonine to replace amino acid number 24 (leucine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing, 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine), 66 (glutamine), 68 (arginine), 81 (iso Leucine), 99 (Asparagine), 100 (Proline), 101 (Valine), 104 (Aspartic Acid), 106 (Isoleucine), 108 (Threonine), 110 (phenylalanine), 122 (alanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 The light chain was named "h#32A1-T7L-type light chain".

The amino acid sequence of the h#32A1-T7L-type light chain is represented by SEQ ID NO: 78 in the Sequence Listing. The sequence comprising amino acid residues 1-113 of the amino acid sequence of SEQ ID NO: 78 and the sequence comprising amino acid residues 114-218 thereof correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 78 is also shown in Figure 40.

c)-viii) h#32A1-T8L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, valine, serine, serine, leucine, glutamic acid, valine, valine , tyrosine, glycine, valine, isoleucine, lysine and threonine to replace amino acid number 24 (leucine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing, 29 (alanine), between 29 and 30 (i.e. insertion of residues deleted in the rat frame), 36 (glutamine), 41 (serine), 66 (glutamine), 81 (isoleucine acid), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (benzene Alanine), 122 (Alanine), 127 (Leucine), 129 (Leucine), 130 (Arginine) and 132 (Alanine) designed humanized #32A1 light chain Named "h#32A1-T8L-type light chain".

The amino acid sequence of the h#32A1-T8 L-type light chain is represented by SEQ ID NO: 79 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 79 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 79 is also shown in Figure 40.

c)-ix) h#32A1-T9L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine , valine, glycine, valine, isoleucine, lysine and threonine to replace amino acid number 24 (leucine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing, 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat frame), 36 (glutamine), 41 (serine), 66 (glutamine), 68 (arginine ), 81 (isoleucine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threo amino acid), 122 (alanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was Named "h#32A1-T9L-type light chain".

The amino acid sequence of the h#32A1-T9L-type light chain is represented by SEQ ID NO: 80 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 80 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 80 is also shown in Figure 40.

c)-x) h#32A1-T10L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine , valine, tyrosine, valine, isoleucine, lysine and threonine replace the amino acid number 24 (leucine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing ), 29 (alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 68 (sperm amino acid), 81 (isoleucine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 The light chain was named "h#32A1-T10L-type light chain".

The amino acid sequence of the h#32A1-T10L-type light chain is represented by SEQ ID NO: 81 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 81 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 81 is also shown in Figure 40.

c)-xi) h#32A1-T11 L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, valine, serine, serine, leucine, glutamic acid, valine, valine, tyrosine , glycine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing acid), between 29 and 30 (i.e., inserting the missing residue in the rat framework), 36 (glutamine), 66 (glutamine), 81 (isoleucine), 99 (asparagine) , 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine), 122 (alanine acid), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T11L -type light chain".

The amino acid sequence of the h#32A1-T11 L-type light chain is represented by SEQ ID NO: 82 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 82 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 82 is also shown in Figure 40.

c)-xii) h#32A1-T12L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine, valine , glycine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing acid), between 29 and 30 (i.e. inserted in the missing residue in the rat framework), 36 (glutamine), 66 (glutamine), 68 (arginine), 81 (isoleucine) , 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 122 (alanine ), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T12L- type light chain".

The amino acid sequence of the h#32A1-T12 L-type light chain is represented by SEQ ID NO: 83 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 83 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 83 is also shown in Figure 41.

c)-xiii) h#32A1-T13 L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine, valine , tyrosine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 ( alanine), between 29 and 30 (i.e. inserting residues missing in the rat framework), 36 (glutamine), 66 (glutamine), 68 (arginine), 81 (isoleucine acid), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (benzene alanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chains were named "h#32A1 -T13L-type light chain".

The amino acid sequence of the h#32A1-T13 L-type light chain is represented by SEQ ID NO: 84 in the Sequence Listing. The sequence comprising amino acid residues 1-113 of the amino acid sequence of SEQ ID NO: 84 and the sequence comprising amino acid residues 114-218 thereof correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 84 is also shown in Figure 41.

c)-xiv) h#32A1-T14 L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, valine, serine, serine, leucine, glutamic acid, valine, valine , glycine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing acid), between 29 and 30 (i.e. inserted in the missing residue in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 81 (isoleucine), 99 (Asparagine), 100 (Proline), 101 (Valine), 104 (Aspartic Acid), 106 (Isoleucine), 108 (Threonine), 122 (Alanine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T14L-type light chain chain".

The amino acid sequence of the h#32A1-T14 L-type light chain is represented by SEQ ID NO: 85 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 85 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 85 is also shown in Figure 41.

c)-xv) h#32A1-T15L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, valine, serine, serine, leucine, glutamic acid, valine, valine , tyrosine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 ( alanine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 81 (isoleucine) , 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine acid), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T15L -type light chain".

The amino acid sequence of the h#32A1-T15L-type light chain is represented by SEQ ID NO: 86 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 86 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 86 is also shown in Figure 41.

c)-xvi) h#32A1-T16L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine , valine, valine, isoleucine, lysine and threonine replace the amino acid numbers 24 (leucine), 29 ( alanine), between 29 and 30 (i.e. inserting the missing residue in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 68 (arginine), 81 (isoleucine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine ), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T16L- type light chain".

The amino acid sequence of the h#32A1-T16 L-type light chain is represented by SEQ ID NO: 87 in the Sequence Listing. The sequence comprising amino acid residues 1-113 of the amino acid sequence of SEQ ID NO: 87 and the sequence comprising amino acid residues 114-218 thereof correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 87 is also shown in Figure 41.

c)-xvii) h#32A1-T17L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, valine, serine, serine, leucine, glutamic acid, valine, valine, glycine, valine amino acid number 24 (leucine), 29 (alanine), 29 of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing, isoleucine, lysine and threonine Between and 30 (i.e. inserting residues deleted in the rat framework), 36 (glutamine), 66 (glutamine), 81 (isoleucine), 99 (asparagine), 100 (proline amino acid), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 122 (alanine), 127 (leucine), 129 ( Leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T17L-type light chain".

The amino acid sequence of the h#32A1-T17 L-type light chain is represented by SEQ ID NO: 88 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 88 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 88 is also shown in Figure 42.

c)-xviii) h#32A1-T18 L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, valine, serine, serine, leucine, glutamic acid, valine, valine, tyrosine , valine, isoleucine, lysine and threonine replace amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing , between 29 and 30 (i.e. inserted in the framework of the deleted residue in the rat), 36 (glutamine), 66 (glutamine), 81 (isoleucine), 99 (asparagine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine), 127 (leucine) , 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chains were named "h#32A1-T18L-type light chain".

The amino acid sequence of the h#32A1-T18 L-type light chain is represented by SEQ ID NO: 89 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 89 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 89 is also shown in Figure 42.

c)-xix) h#32A1-T19L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, proline, lysine, valine, serine, serine, leucine, glutamic acid, valine, valine , valine, isoleucine, lysine and threonine replace amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing , between 29 and 30 (i.e. inserted in the rat framework deleted residues), 36 (glutamine), 66 (glutamine), 68 (arginine), 81 (isoleucine), 99 (Asparagine), 100 (Proline), 101 (Valine), 104 (Aspartic Acid), 106 (Isoleucine), 108 (Threonine), 127 (Leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T19L-type light chain".

The amino acid sequence of the h#32A1-T19 L-type light chain is represented by SEQ ID NO: 90 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 90 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 90 is also shown in Figure 42.

c)-xx) h#32A1-T20L-type light chain:

By using methionine, aspartic acid, serine, glutamic acid, asparagine, proline, valine, serine, serine, leucine, glutamic acid, valine, valine , valine, isoleucine, lysine and threonine replace amino acid numbers 24 (leucine), 29 (alanine) of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing , between 29 and 30 (i.e. inserting residues deleted in the rat framework), 36 (glutamine), 41 (serine), 66 (glutamine), 81 (isoleucine), 99 (day Paragine), 100 (proline), 101 (valine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 127 (leucine), 129 ( Leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T20L-type light chain".

The amino acid sequence of the h#32A1-T20L-type light chain is represented by SEQ ID NO: 91 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 91 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 91 is also shown in Figure 42.

c)-xxi) h#32A1-T21L-type light chain:

By using glutamic acid, methionine, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, glutamic acid, Proline, glutamic acid, phenylalanine, leucine, tyrosine, glycine, glutamine, valine, isoleucine, lysine and threonine are replaced by SEQ in the sequence listing Amino acid numbers 21 (aspartic acid), 24 (leucine), 29 and 30 of the light chain of #32A1 represented by ID NO: 30 (i.e. inserted in the residue missing in the rat framework), 31 (alcohol amino acid), 32 (valine), 34 (leucine), 36 (glutamine), 40 (isoleucine), 66 (glutamine), 99 (asparagine), 100 (proline amino acid), 101 (valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 ( phenylalanine), 122 (alanine), 123 (glycine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) The h#32A1 light chain was named "h#32A1-T21L-type light chain".

The amino acid sequence of the h#32A1-T21 L-type light chain is represented by SEQ ID NO: 92 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 92 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 92 is also shown in Figure 42.

c)-xxii) h#32A1-T22L-type light chain:

By using glutamic acid, methionine, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, glutamic acid, Proline, glutamic acid, phenylalanine, leucine, glutamine, valine, isoleucine, lysine and threonine are replaced by SEQ ID NO: 30 in the sequence listing Amino acid numbers 21 (aspartic acid), 24 (leucine), 29 and 30 of the light chain of #32A1 (i.e. insertion of residues missing in the rat framework), 31 (alanine), 32 ( valine), 34 (leucine), 36 (glutamine), 40 (isoleucine), 66 (glutamine), 99 (asparagine), 100 (proline), 101 ( valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 123 (glycine), 127 (leucine amino acid), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1-T22L-type light chain".

The amino acid sequence of the h#32A1-T22 L-type light chain is represented by SEQ ID NO: 93 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 93 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 93 is also shown in Figure 43.

c)-xxiii) h#32A1-T23L-type light chain:

By using methionine, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, glutamic acid, proline, Glutamic acid, phenylalanine, leucine, valine, isoleucine, lysine and threonine replace amino acid number 24 of #32A1 light chain represented by SEQ ID NO: 30 in the sequence listing (leucine), between 29 and 30 (i.e. insertion of residues missing in the rat framework), 31 (alanine), 32 (valine), 34 (leucine), 36 (glutamine amide), 40 (isoleucine), 66 (glutamine), 99 (asparagine), 100 (proline), 101 (valine), 102 (glutamine), 103 (alanine acid), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine Amino acid) designed humanized #32A1 light chain was named "h#32A1-T23L-type light chain".

The amino acid sequence of the h#32A1-T23 L-type light chain is represented by SEQ ID NO: 94 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 94 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 94 is also shown in Figure 43.

c)-xxiv) h#32A1-T24L-type light chain:

By using glutamic acid, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, glutamic acid, proline, glutamic acid Amino acid, phenylalanine, leucine, tyrosine, glycine, glutamine, valine, isoleucine, lysine and threonine substitutions are represented by SEQ ID NO: 30 in the sequence listing Amino acid numbers 21 (aspartic acid), 29 and 30 of the light chain of #32A1 (i.e. insertion of residues missing in the rat frame), 31 (alanine), 32 (valine), 34 (Leucine), 36 (Glutamine), 40 (Isoleucine), 66 (Glutamine), 99 (Asparagine), 100 (Proline), 101 (Valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine), 122 (alanine) , 123 (glycine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h# 32A1-T24L-Type Light Chain".

The amino acid sequence of the h#32A1-T24 L-type light chain is represented by SEQ ID NO: 95 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 95 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 95 is also shown in Figure 43.

c)-xxv) h#32A1-T25L-type light chain:

By using threonine, serine, leucine, proline, glutamic acid, leucine, serine, serine, leucine, glutamic acid, proline, glutamic acid, phenylalanine, Tyrosine, valine, isoleucine, lysine and threonine are substituted by SEQ ID NO: 30 in the sequence listing between amino acid numbers 29 and 30 of #32A1 light chain (i.e. inserted between large murine framework), 31 (alanine), 32 (valine), 34 (leucine), 36 (glutamine), 40 (isoleucine), 99 (asparagine ), 100 (proline), 101 (valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 110 (phenylalanine amino acid), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized #32A1 light chain was named "h#32A1- T25L-Type Light Chain".

The amino acid sequence of the h#32A1-T25L-type light chain is represented by SEQ ID NO: 96 in the Sequence Listing. The sequence comprising amino acid residues 1-113 and the sequence comprising amino acid residues 114-218 of the amino acid sequence of SEQ ID NO: 96 correspond to the light chain variable region and the light chain constant region, respectively. The amino acid sequence of SEQ ID NO: 96 is also shown in Figure 43.

Example 23

Preparation of humanized antibody gene of rat anti-mouse Siglec-15 monoclonal antibody #32A1

a) Construction of h#32A1-T1L, h#32A1-T2L, h#32A1-T3L, h#32A1-T4L, h#32A1-T5L and h#32A1-T6L-type light chain expression vectors

DNAs were synthesized (Medical & Biological Laboratories Co., Ltd., artificial gene synthesis service) each containing amino acids encoded by amino acid numbers 1-133, SEQ ID NO: 63, respectively, of SEQ ID NO: 61 in the Sequence Listing Amino acid number 1-133, amino acid number 1-133 of SEQ ID NO: 65, amino acid number 1-132 of SEQ ID NO: 67, amino acid number 1-133 of SEQ ID NO: 69 or amino acid number 1 of SEQ ID NO: 71 h#32A1-T1L, h#32A1-T2L, h#32A1-T3L, h#32A1-T4L, h#32A1-T5L or h#32A1-T6L-type light chain variable fused to secretion signal represented by -133 region genes. Then, the synthesized DNA was cut with restriction enzymes NheI and BsiWI, and each DNA fragment obtained was inserted into a universal humanized antibody light chain expression vector (pEF6KCL) at the site cut with restriction enzymes NheI and BsiWI, thereby constructing h#32A1-T1L, h#32A1-T2L, h#32A1-T3L, h#32A1-T4L, h#32A1-T5L and h#32A1-T6L-type light chain expression vectors. The expression vectors thus obtained were named "pEF6KCL/h#32A1-T1L", "pEF6KCL/h#32A1-T2L", "pEF6KCL/h#32A1-T3L", "pEF6KCL/h#32A1-T4L", " pEF6KCL/h#32A1-T5L" and "pEF6KCL/h#32A1-T6L".

b) Construction of h#32A1-T1H, h#32A1-T2H, h#32A1-T3H, h#32A1-T5H and h#32A1-T6H heavy chain expression vectors

DNAs were synthesized (Medical & Biological Laboratories Co., Ltd., artificial gene synthesis service) each containing amino acid numbers 1-140 encoded by SEQ ID NO: 51, 53, 55, 57 or 59 in the Sequence Listing Genes for h#32A1-T1H, h#32A1-T2H, h#32A1-T3H, h#32A1-T5H or h#32A1-T6H-type heavy chain variable regions fused to secretion signals are indicated. Then, the synthesized DNA was cut with restriction enzyme BlpI, and each DNA fragment obtained was inserted into a general-purpose humanized antibody heavy chain expression vector (pEF1/FCCU-1) at the site cut with restriction enzyme BlpI, thereby constructing h#32A1-T1H, h#32A1-T2H, h#32A1-T3H, h#32A1-T5H and h#32A1-T6H heavy chain expression vectors. The expression vectors thus obtained were named "pEF1/FCCU/h#32A1-T1H", "pEF1/FCCU/h#32A1-T2H", "pEF1/FCCU/h#32A1-T3H", "pEF1/FCCU/ h#32A1-T5H" and "pEF1/FCCU/h#32A1-T6H".

Example 24

Preparation of humanized antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1

a) Preparation of humanized antibody

Inoculate 1.5 x 10 ⁸ cells of 293 FreeStyle cells in the logarithmic growth phase into 100 ml fresh FreeStyle 293 expression medium (Invitrogen, Inc.), and culture in an 8% CO ₂ incubator at 37ºC with shaking ( 125 rpm). 1 mg of polyethyleneimine (manufactured by Polyscience, Inc. #24765) was dissolved in 4 ml of Opti-Pro SFM medium (manufactured by Invitrogen, Inc.), and the resulting solution was left at room temperature for 5 minutes. A heavy chain expression plasmid (0.05 mg) and a light chain expression plasmid (0.15 mg) prepared using PureLink HiPure Plasmid Kit (Invitrogen, Inc.) were suspended in 4 ml of Opti-Pro SFM medium (Invitrogen, Inc.). Then, 4 ml of the resulting expression plasmid/Opti-Pro SFM mixture was added to 4 ml of the polyethyleneimine/Opti-Pro SFM mixture that had been left at room temperature for 5 minutes, and the resulting mixture was left at room temperature for another 5 minutes. Then, add 8 ml of polyethyleneimine/expression plasmid/Opti-Pro SFM mixture into the 293 FreeStyle cell suspension, and continue shaking culture. After culturing the cells for 7 days at 37ºC in 8% CO ₂ , collect the culture supernatant.

b) Purification of humanized antibodies

The culture supernatant obtained in a) above was purified by protein A affinity chromatography. 100 ml of the culture supernatant was put into a 500-ml flask with a stopper, and 1 ml of a suspension (50% slurry) of MabSelect SuRe (manufactured by GE Healthcare Bio-science Co., Ltd.) equilibrated with PBS was added thereto . The resulting mixture was stirred overnight in a 10 ºC incubator at 100 rpm. Then, the 293F culture supernatant/MabSelect SuRe suspension was loaded onto a Zeba Spin empty cartridge 5 mL (PIERCE, Inc.). After pouring all the resin into the column, wash the column with 10 ml of 1 M NaCl. Subsequently, 1 ml of 1 M arginine solution (pH 4.0) was loaded onto the column and the antibody-containing fractions were collected. This fraction was added to a centrifugal filter unit (Amicon Ultra-4, fractional molecular weight: 50K, Millipore Co., Ltd.), the liquid was replaced with a citrate buffer, and concentrated. The final volume was brought up to 200 μl and used as purified sample.

The humanized antibody of #32A1 obtained from the combination of pEF6KCL/h#32A1-T1L and pEF1/FCCU/h#32A1-T1H was named "h#32A1-T1"; The humanized antibody of #32A1 obtained from the combination of /FCCU/h#32A1-T2H was named "h#32A1-T2"; from the combination of pEF6KCL/h#32A1-T3L and pEF1/FCCU/h#32A1-T3H The obtained humanized antibody of #32A1 was named "h#32A1-T3"; the humanized antibody of #32A1 obtained from the combination of pEF6KCL/h#32A1-T4L and pEF1/FCCU/h#32A1-T3H was named Named "h#32A1-T4"; the #32A1 humanized antibody obtained from the combination of pEF6KCL/h#32A1-T5L and pEF1/FCCU/h#32A1-T5H was named "h#32A1-T5"; And the humanized antibody of #32A1 obtained from the combination of pEF6KCL/h#32A1-T6L and pEF1/FCCU/h#32A1-T6H was named "h#32A1-T6".

Example 25

Evaluation of the Binding Properties of Rat Anti-Mouse Siglec-15 Monoclonal Antibody #32A1 Humanized Antibody to Mouse Siglec-15 Protein

The binding properties of rat anti-mouse Siglec-15 #32A1 T1-T6 humanized antibodies to mouse Siglec-15-Fc and human Siglec-15-Fc were evaluated by the following method. Using PBS, the mouse Siglec-15-Fc purified in Example 4 or the human Siglec-15-Fc purified in Example 12 was diluted to 1 μg/ml, and the diluted solution was dispensed at 100 μl/well To the immunoplate (manufactured by Nunc, Inc., #437111), let the plate stand overnight at 4ºC, thereby allowing each protein to adsorb to the plate. The next day, wash each well 5 times with PBS-T solution (PBS, 0.05% (v/v) Tween 20), and distribute skim milk (Morinaga Milk Industry Co., Ltd.) diluted with PBS at 350 μl/well. . production) to 5% of the resulting solution and let the plate stand at room temperature for 2 hours. The liquid in each well was removed, and each well was washed 5 times with PBS-T solution. Thereafter, each of the purified rat anti-mouse Siglec-15 #32A1 T1 to T6 humanized antibodies prepared in Example 24 or prepared in Example 19 was purified using a PBS solution containing 0.5% skim milk. The human chimeric antibody was diluted to a final concentration of 1-0.004 μg/ml (4-fold dilution series), at 100 μl/well, the diluted solution was dispensed, and the plate was left to stand at room temperature for 2 hours. At this time, the concentration of each antibody was determined by measuring at 280 nm using a spectrophotometer DU-7400 (manufactured by Beckman Coulter, Inc.), and calculating based on the molar molecular extinction coefficient of each antibody. After each well had been washed 5 times with PBS-T solution, basic phosphoric acid diluted to 2500-fold with TBS-T solution (TBS, 0.05% (v/v) Tween 20) was added at 100 μl/well. Enzyme-conjugated AffiniPure goat anti-human IgG (manufactured by Jackson ImmunoResearch, Inc., #109-055-097), and the plate was left to stand at room temperature for 1 hour. The liquid in each well was removed, and each well was washed 5 times with TBS-T solution. Thereafter, a fluorescent substrate solution (manufactured by Roche Co., Ltd., #11681982001) was added at 100 μl/well, and a fluorescent reaction was performed. 10 minutes after the addition of the fluorescent substrate solution (in the case of plates with mouse Siglec-15-Fc adsorbed thereto) and 15 minutes after (in the case of plates with human Siglec-15-Fc adsorbed thereto) , and the fluorescence intensity was measured using SpectraMax M5 (manufactured by Molecular Devices, Inc.). The results confirmed: all 6 kinds of rat anti-mouse Siglec-15 humanized antibodies were combined with mouse Siglec-15 protein (Fig. 18) and human Siglec-15 protein (Fig. 19) in an antibody concentration-dependent manner. ).

Example 26

Effect of adding rat anti-mouse Siglec-15 monoclonal antibody #32A1 chimeric antibody on osteoclast differentiation (stimulated by TNFα) of mouse bone marrow non-adherent cells

Using the rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 6 and the chimeric antibody of the rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 9, check Effects on osteoclast differentiation of mouse bone marrow non-adherent cells stimulated by TNFα. With α-MEM medium containing 10% fetal bovine serum (FBS), 10 ng/ml M-CSF and 2 ng/ml TGF-β (manufactured by R&D Systems, Inc.), the small Rat bone marrow non-adherent cells were prepared at 1.5×10 ⁵ cells/ml, and the obtained cell preparation was seeded in each well of a 96-well plate in an amount of 200 μl, and the cells were cultured in a CO ₂ incubator for 2 days. The old culture solution in the 96-well plate was removed, and 100 μl of MEM-α medium was added to each well. The 100 μl of MEM-α medium contained 10% FBS to which recombinant human TNFα (manufactured by R&D Systems, Inc.) had been added. ) and M-CSF, the final concentrations were 30 ng/ml and 10 ng/ml, respectively. To this cell culture medium, the rat anti-mouse Siglec-15 monoclonal antibody #32A1 prepared in Example 6 or the rat anti- - Chimeric antibody to mouse Siglec-15 monoclonal antibody #32A1, cells were cultured for an additional 3 days in a CO ₂ incubator. At the same time, a well was also prepared in which recombinant human OCIF/OPG prepared by the method described in the specification of Patent Application Publication No. WO 96/26217 was added at a concentration of 100 ng/ml for cell culture. After the culture was completed, TRAP staining was performed using a leukocyte acid phosphatase kit (manufactured by Sigma Co., Ltd.) according to the instructions attached to the kit, and the formation of TRAP-positive multinucleated osteoclasts was observed. As a result, the addition of anti-mouse Siglec-15 polyclonal antibody inhibited the formation of TRAP-positive giant multinucleated osteoclasts (Fig. 20). Although small mononuclear osteoclasts were formed (even with the addition of rat anti-mouse Siglec-15 mAb #32A1 or its chimeric antibody), formation of multinucleated osteoclasts induced by TNFα was almost complete suppressed. On the other hand, even when a sufficient amount of OCIF/OPG, which is a RANKL blocker, was added, the formation of multinucleated osteoclasts was only slightly inhibited. Furthermore, in the same manner, by the method described in Example 9, the activity of tartrate-resistant acid phosphatase (TRAP) of the formed osteoclasts was measured. After the enzymatic reaction was stopped, the absorbance at 405 nm of each well was measured, and the resulting measurement was used as an index of TRAP activity. The result is shown in Figure 21. Significant inhibition of TRAP activity was observed with #32A1 antibody and #32A1 chimeric antibody in the range of 125 ng/ml to 4 μg/ml. These results show that rat anti-mouse Siglec-15 monoclonal antibody and its chimeric antibody strongly inhibit the cell fusion process in differentiation and maturation of osteoclasts induced by TNFα.

Example 27

Evaluation of the biological activity of rat anti-mouse Siglec-15 monoclonal antibodies #8A1 and #32A1 based on a rat osteoclastogenesis assay

Using the rat anti-mouse Siglec-15 monoclonal antibodies #8A1 and #32A1 prepared in Example 6, the effect on osteoclast differentiation of rat bone marrow non-adherent cells was examined. By improving the method in Example 8, non-adherent rat bone marrow cells were prepared. That is, femurs were excised from both legs of 12-week-old female F344 rats, and soft tissues were removed. Cut off both ends of the femur, inject MEM-α medium with a syringe with a 23-gauge injection needle to push out the bone marrow cells, and collect them in a centrifuge tube. Centrifuge at 100 g for 5 minutes at room temperature and remove the supernatant. 1 ml of hemolysis buffer (erythrocyte lysis buffer, manufactured by Sigma Co., Ltd.) was added to the obtained cell pellet for suspension, and the obtained suspension was left at room temperature for 5 minutes. 20 ml of D-PBS was added thereto, and the suspension was centrifuged at 100 g for 5 minutes at room temperature, and the supernatant was removed. To the obtained cell pellet was added 10 ml of MEM-α medium (manufactured by Invitrogen, Inc.) containing 5 ng/ml M-CSF (manufactured by R&D Systems, Inc.) and 10% fetal bovine serum (FBS) to mix. hanging. Then, the resulting suspension was passed through a cell strainer (40 μm nylon, produced by BD Falcon) to remove aggregates. The resulting cells were transferred to a 75 cm ² T-flask (for attachment of adherent cells) and cultured overnight in a CO ₂ incubator. After culturing overnight, cells not attached to the T-flask were collected and used as rat bone marrow non-adherent cells.

Rat bone marrow non-adherent cells prepared by this method were cultured, and a rat osteoclast formation test was performed according to the method of Example 9. That is, in α-MEM medium containing 10% FBS and 10 ng/ml M-CSF (manufactured by R&D Systems, Inc.), the rat bone marrow non-adherent cells thus prepared were made 1.5×10 ⁵ cells/ml, and inoculate 200 μl of the obtained cell preparation into each well of a 96-well plate, and culture the cells in a CO ₂ incubator for 2 days. The old culture solution in the 96-well plate was removed, and 100 μl of MEM-α medium containing 10% FBS to which human RANKL (RANKL, produced by Peprotech, Inc.) had been added was added to each well. ) and M-CSF, the final concentrations were 20ng/ml and 10ng/ml, respectively. To this cell culture medium, add the rat anti-mouse Siglec-15 monoclonal antibody #8A1 or #32A1 prepared in Example 6 at a concentration of 31-1000 ng/ml, and culture in a _CO incubator Cells for an additional 3 days. After the end of the culture, the tartrate-resistant acid phosphatase (TRAP) activity of the formed osteoclasts was measured by the method described in Example 9. After the enzymatic reaction was stopped, the absorbance at 405 nm of each well was measured, and the resulting measurement was used as an index of TRAP activity. The result is shown in Figure 22. In the case of #8A1 antibody, dose-dependent inhibition of TRAP activity was observed at a concentration of 63 ng/ml or higher, and in the case of #32A1 antibody, at a concentration of 31 ng/ml or higher, A dose-dependent inhibition of TRAP activity was observed. These results show that rat anti-mouse Siglec-15 monoclonal antibodies #8A1 and #32A1 strongly inhibited rat osteoclast formation in the same manner as mouse osteoclast formation, and that #32A1 antibody inhibited rat osteoclast formation. The in vitro activity of osteoclast formation was higher than that of #8A1 antibody.

Example 28

Humanized Antibody Design of Rat Anti-Mouse Siglec-15 Monoclonal Antibody #32A1 (2)

a) Design of humanized form of #32A1

a)-i) Design of the amino acid sequence of humanized #32A1

Humanized #32A1 antibody was constructed according to a method generally called CDR grafting (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). Regarding the definition of the CDR region, all except CDRH2 are defined by Kabat (SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST VOL. I, 5th edition (1991)). Regarding CDRH2, using its own definition, its sequence is shortened by 5 residues at the C-terminus than the Kabat definition. In the heavy chain sequence containing CDRH2 thus defined, the rat-derived CDR sequence is made shorter and incorporates more human framework sequences, so when it is administered to humans, it is recognized as a xenoantigen Less likely. Recipient antibodies are selected based on amino acid homology within the framework regions. The framework region sequence of #32A1 was compared with all human framework sequences in the Kabat database (Nuc. Acid Res. 29, 205-206 (2001)) containing antibody amino acid sequences. As a result, for the L-chain, GF4/1.1'CL was selected as the acceptor based on the sequence homology of 71% in the framework region. For the H-chain, M37GO37'CL was chosen as the acceptor based on the 73% sequence homology in the framework regions. The amino acid residues in the framework region of GF4/1.1'CL (for the L-chain) and the framework region of M37GO37'CL (for the H-chain) were compared with those of #32A1 to identify Show the positions where different amino acids are used. Using the three-dimensional model of #32A1 constructed in a)-i) of Example 22, the positions of these residues were analyzed. The donor residues to be grafted onto the recipient are then selected according to the criteria provided by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989)). The humanized #32A1 sequence was constructed as described in the Examples below by transferring some selected donor residues to the recipient antibody.

a)-ii) Humanization of #32A1 heavy chain

a)-ii)-i) h#32A1-H1-1-type heavy chain:

By separately using leucine, valine, serine, glutamine, alanine, glycine, alanine, valine, lysine, phenylalanine, asparagine, alanine, lysine acid, asparagine, leucine, methionine, asparagine, valine, alanine, threonine, leucine and serine were substituted for 32A1 represented by SEQ ID NO: 28 in the Sequence Listing Amino acid number 23 (isoleucine), 24 (leucine), 26 (threonine), 32 (lysine), 43 (threonine), 61 (glutamic acid), 83 (glutamine acid), 85 (leucine), 86 (glutamic acid), 89 (valine), 95 (aspartic acid), 96 (serine), 97 (glutamic acid), 98 (serine), 100 (valine), 104 (valine), 105 (serine), 114 (isoleucine), 118 (threonine), 134 (valine), 135 (methionine) and 140 ( Leucine) designed humanized 32A1 heavy chain was named "h#32A1-H1-1-type heavy chain".

The amino acid sequence of the h#32A1-H1-1-type heavy chain is represented by SEQ ID NO: 99 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQID NO: 99, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-466 correspond to the signal sequence, heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 99 is represented by SEQ ID NO: 98 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 98, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1398 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 98 and the amino acid sequence of SEQ ID NO: 99 are also shown in Figure 54.

a)-iii) Humanization of #32A1 light chain

a)-iii)-i) h#32A1-L2-15-type light chain:

By using glutamic acid, leucine, methionine, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, Glutamic acid, proline, glutamic acid, phenylalanine, leucine, tyrosine, glycine, glutamine, valine, isoleucine, lysine and threonine are replaced by sequence Between the amino acid numbers 21 (aspartic acid), 23 (valine), 24 (leucine), 29 and 30 of 32A1 represented by SEQ ID NO:30 in the list (i.e. the insertion is deleted in the rat framework ), 31 (alanine), 32 (valine), 34 (leucine), 36 (glutamine), 40 (isoleucine), 66 (glutamine), 99 ( asparagine), 100 (proline), 101 (valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 110 (phenylalanine), 122 (alanine), 123 (glycine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine Amino acid) designed humanized 32A1 light chain was named "h#32A1-L2-15-type light chain".

The amino acid sequence of the h#32A1-L2-15-type light chain is represented by SEQ ID NO: 103 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQID NO: 103, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 103 is represented by SEQ ID NO: 102 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 102, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain variable region sequence and light chain constant region sequence. The nucleotide sequence of SEQ ID NO: 102 and the amino acid sequence of SEQ ID NO: 103 are also shown in Figure 56.

a)-iii)-ii) h#32A1-L2-16-type light chain:

By using glutamic acid, leucine, methionine, threonine, serine, leucine, proline, glutamic acid, leucine, alanine, serine, serine, leucine, Glutamic acid, proline, glutamic acid, phenylalanine, leucine, glutamine, valine, isoleucine, lysine and threonine are replaced by SEQ ID NO in the sequence listing : Amino acid number 21 (aspartic acid), 23 (valine), 24 (leucine), 29 and 30 of 32A1 represented by 30 (i.e. inserted in the missing residue in the rat framework), 31 (alanine), 32 (valine), 34 (leucine), 36 (glutamine), 40 (isoleucine), 66 (glutamine), 99 (asparagine), 100 (proline), 101 (valine), 102 (glutamine), 103 (alanine), 104 (aspartic acid), 106 (isoleucine), 108 (threonine), 123 (glycine), 127 (leucine), 129 (leucine), 130 (arginine) and 132 (alanine) designed humanized 32A1 light chains were named "h#32A1- L2-16-type light chain".

The amino acid sequence of the h#32A1-L2-16-type light chain is represented by SEQ ID NO: 105 in the Sequence Listing. The sequence comprising amino acid residues 1-20 of the amino acid sequence of SEQID NO: 105, the sequence comprising its amino acid residues 21-133 and the sequence comprising its amino acid residues 134-238 correspond to the signal sequence, light chain variable region, respectively and light chain constant regions. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 105 is represented by SEQ ID NO: 104 in the Sequence Listing. The sequence comprising nucleotides 1-60 of the nucleotide sequence of SEQ ID NO: 104, the sequence comprising its nucleotides 61-399 and the sequence comprising its nucleotides 400-714 encode signal sequence, light chain variable region sequence and light chain constant region sequence. The nucleotide sequence of SEQ ID NO: 104 and the amino acid sequence of SEQ ID NO: 105 are also shown in Figure 57.

b) Preparation of humanized antibody gene of rat anti-mouse Siglec-15 monoclonal antibody #32A1

b)-i) Construction of general human heavy chain expression vector pEG2

A DNA fragment (Medical & Biological Laboratories Co., Ltd., artificial gene synthesis service) containing the DNA sequence represented by SEQ ID NO: 106 encoding the signal sequence and the amino acids of the constant region of human IgG2 was synthesized, And it was digested with restriction enzymes NheI and PmeI, thereby obtaining a DNA fragment of about 1100 base pairs. This DNA fragment was ligated to a DNA fragment of about 6200 bp obtained by digesting pEF1/FCCU-1 with NheI and PmeI and removing a DNA fragment of about 1100 bp, thereby constructing a universal humanized antibody (hIgG2 type ) heavy chain expression vector pEG2.

b)-ii) Construction of h#32A1-H1-1 heavy chain expression vector

By synthesizing DNA (Invitrogen) containing a gene encoding the h#32A1-H1-1 heavy chain variable region fused to a secretion signal represented by amino acid numbers 1-140 of SEQ ID NO: 99 in the Sequence Listing, The DNA was cut with restriction enzyme BlpI to obtain a DNA fragment, and the DNA fragment was inserted into a general-purpose humanized antibody (hIgG2 type) heavy chain expression vector (pEG2) at the site cut with restriction enzyme BlpI, thereby constructing h# 32A1-H1-1 heavy chain expression vector. The expression vector thus obtained was named "pEG2/h#32A1-H1-1".

b)-iii) Construction of h#32A1-H5 heavy chain expression vector

The humanized 32A1 heavy chain in which the signal sequence and constant region of the h#32A1-T5H-type heavy chain designed in b)-iv) of Example 22 were changed to the corresponding sequence of IgG2 type was named "h #32A1-H5-type heavy chain".

The amino acid sequence of the h#32A1-H5-type heavy chain is represented by SEQ ID NO: 101 in the Sequence Listing. The sequence comprising amino acid residues 1-19 of the amino acid sequence of SEQ ID NO: 101, the sequence comprising its amino acid residues 20-140 and the sequence comprising its amino acid residues 141-466 correspond to the signal sequence, heavy chain variable region, respectively and heavy chain constant region. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 101 is represented by SEQ ID NO: 100 in the Sequence Listing. The sequence comprising nucleotides 1-57 of the nucleotide sequence of SEQ ID NO: 100, the sequence comprising its nucleotides 58-420 and the sequence comprising its nucleotides 421-1398 respectively encode a signal sequence, a heavy chain can Variable region sequences and heavy chain constant region sequences. The nucleotide sequence of SEQ ID NO: 100 and the amino acid sequence of SEQ ID NO: 101 are also shown in FIG. 55 .

The pEF1/FCCU/h#32A1-T5H prepared in Example 23 was cut with restriction enzyme BlpI to obtain a DNA fragment, which was inserted into a general-purpose humanized antibody (hIgG2 type) recombinant at the site cut with restriction enzyme BlpI. chain expression vector (pEG2), thus constructing h#32A1-H5 heavy chain expression vector. The expression vector thus obtained was named "pEG2/h#32A1-H5".

b)-iv) Construction of h#32A1-L2-15 and h#32A1-L2-16-type light chain expression vectors

Synthetic DNAs (Invitrogen) each containing h#32A1 encoding h#32A1 fused to a secretion signal represented by amino acid numbers 1-133 of SEQ ID NO: 103 or amino acid numbers 1-133 of SEQ ID NO: 105 in the Sequence Listing -L2-15 or h#32A1-L2-16-type light chain variable region gene, and in the same manner as in Example 23, construct h#32A1-L2-15 and h#32A1-L2-16-type Light chain expression vector. The expression vectors thus obtained were named "pEF6KCL/h#32A1-L2-15" and "pEF6KCL/h#32A1-L2-16", respectively.

c) Preparation of humanized antibody of rat anti-mouse Siglec-15 monoclonal antibody #32A1

c)-i) Preparation of humanized antibodies

Inoculate 1.2 x ¹⁰⁹ cells of FreeStyle 293F cells (Invitrogen) in logarithmic growth phase into 1.2 L of fresh FreeStyle 293 Expression Medium (Invitrogen) and shake in an 8% _CO2 incubator at 90 rpm at 37ºC Incubate for 1 hour. 3.6 mg polyethyleneimine (Polyscience, #24765) was dissolved in 20 ml Opti-Pro SFM medium (Invitrogen). Subsequently, H-chain expression plasmid (0.4 mg) and L-chain expression plasmid (0.8 mg) prepared using PureLink HiPure Plasmid Kit (Invitrogen, Inc.) were suspended in 20 ml of Opti-Pro SFM medium. Then, 20 ml of the resulting expression plasmid/Opti-Pro SFM mixture was added to 20 ml of polyethyleneimine/Opti-Pro SFM mixture, and the resulting mixture was gently stirred and then left for 5 minutes. Thereafter, the mixture was added to FreeStyle 293F cells and cultured in an 8% CO ₂ incubator at 37 ºC with shaking at 90 rpm for 7 days. The resulting culture supernatant was filtered through a disposable capsule filter (Advantec #CCS-045-E1H).

The humanized antibody of #32A1 obtained from the combination of pEF6KCL/h#32A1-T5L and pEG2/h#32A1-H5 was named "h#32A1-H5/L5"; The humanized antibody of #32A1 obtained by the combination of /h#32A1-H1-1 was named "h#32A1-H1-1/L5"; pEF6KCL/h#32A1-L2-15 and pEG2/h#32A1 -The humanized antibody of #32A1 obtained from the combination of H1-1 was named "h#32A1-H1-1/L2-15"; and pEF6KCL/h#32A1-L2-16 and pEG2/h#32A1- The #32A1 humanized antibody obtained from the combination of H1-1 was named "h#32A1-H1-1/L2-16".

c)-ii) Purification of humanized antibodies

The culture supernatant obtained in c)-i) above was purified by a 2-step method involving rProtein A affinity chromatography (4-6ºC) and ceramic hydroxyapatite (at room temperature). After rProtein A affinity chromatography purification and after ceramic hydroxyapatite purification, a buffer exchange step was performed at room temperature. First, 1100-1200 ml of culture supernatant was loaded onto MabSelect SuRe (manufactured by GE Healthcare Bioscience Co., Ltd., 2 x 1 ml HiTrap columns in series) equilibrated with PBS. After pouring all the medium into the column, wash the column with 15-30 ml PBS. Subsequently, elution was performed with a 2 M arginine hydrochloride solution (pH 4.0), and antibody-containing fractions were collected. The fractions were loaded onto a desalting column (manufactured by GE Healthcare Bioscience Co., Ltd., 2 x 5 ml HiTrap desalting columns in series), and washed with a buffer (pH 6.5) containing 5 mM sodium phosphate, 50 mM MES, and 20 mM NaCl Replacement fluid. In addition, the buffer-exchanged antibody solution was loaded onto a ceramic hydroxyapatite column (Japan Bio-Rad Laboratories, Inc., Bio-ScaleCHT2-1 hydroxyapatite column (2 ml volume)) containing Equilibrate in a buffer (pH 6.5) of 5 mM NaPi, 50 mM MES, and 20 mM NaCl. Then, linear concentration gradient elution was performed using sodium chloride, and antibody-containing fractions were collected. The fractions were loaded onto a desalting column (manufactured by GE Healthcare Bioscience Co., Ltd., 2 x 5 ml HiTrap desalting columns in series), and washed with CBS (10 mM citrate buffer containing 140 mM sodium chloride, pH 6.0) Replacement fluid. Finally, using a centrifugal ultrafiltration (UF) filter unit VIVASPIN 20 (separation molecular weight: 30 K, Sartorius Co., Ltd., at 4ºC), the resulting solution was concentrated to adjust the concentration of IgG to 1.0 mg/ml or higher , and the thus obtained solution was used as a purified sample. The volumes of the various purified samples were as follows: h#32A1-H1-1/L5: 2.2 ml, h#32A1-H5/L5: 1.8 ml, h#32A1-H1-1/L2-16: 6.0 ml, and h#32A1-H1-1/L2-16: 6.0 ml, and h #32A1-H1-1/L2-15: 2.2 ml.

Example 29

Evaluation of the biological activity of the humanized antibody of rat anti-mouse Siglec-15 mAb #32A1 based on the mouse osteoclast formation assay

By partially modifying the method described in Example 9, the inhibitory effect on mouse osteoclastogenesis exhibited by the humanized antibodies obtained in Examples 24 and 28 was evaluated. In α-MEM medium containing 10% FBS and 10 ng/ml M-CSF (manufactured by R&D Systems, Inc.), at ^1.5 x 105 cells/ml, the mouse prepared by the method of Example 8 was prepared Bone marrow non-adherent cells, the resulting cell preparation was inoculated into each well of a 96-well plate in an amount of 200 μl, and the cells were cultured in a CO ₂ incubator for 2 days. The old culture solution in the 96-well plate was removed, and 100 μl of MEM-α medium was added to each well. The 100 μl of MEM-α medium contained 10% FBS to which human RANKL (RANKL, Peprotech, Inc.) and M-CSF at a final concentration of 20 ng/ml and 10 ng/ml, respectively. To this cell culture solution, the rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) prepared in Example 6 or the anti-mouse Siglec-15 monoclonal antibody prepared in Example 24 was added at a concentration of 3-100 ng/ml. Each of the 6 classes of humanized #32A1 antibodies (h#32A1-T1, h#32A1-T2, h#32A1-T3, h#32A1-T4, h#32A1-T5 and h#32A1-T6 ), and culture the cells for an additional 3 days in a _CO incubator. After the end of the culture, the activity of tartrate-resistant acid phosphatase (TRAP) of the formed osteoclasts was measured by the following method. The culture medium in each well of the 96-well plate was removed by aspiration, and 50 μl of 50 mM sodium citrate buffer (pH 6.1) containing 1% Triton X-100 was added to each well. Then, shake the plate on a plate shaker for 5 minutes to lyse the cells. Add 50 μl of substrate solution (50 mM sodium citrate buffer (pH 6.1) containing 5 mg/ml p-nitrophenyl phosphate and 0.46% sodium tartrate) to each well, and incubate the plate at room temperature. Incubate for 10 minutes. After incubation, 50 µl of 1N sodium hydroxide solution was added to each well of the 96-well plate to stop the enzyme reaction. After the enzymatic reaction was stopped, the absorbance at 405 nm of each well was measured, and the resulting measurement was used as an index of TRAP activity. The results are shown in Figures 44 and 45. In addition, for the rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) prepared in Example 6, the human chimeric antibody of #32A1 antibody prepared in Example 19, and in Example 28 Four types of humanized #32A1 antibodies (h#32A1-H1-1/L5, h#32A1-H5/L5, h#32A1-H1-1/L2-16 and h#32A1-H1-1/ L2-15), the biological activity was evaluated by the same method as this test, and the results are shown in FIGS. 46 and 47 . 10 types of humanized #32A1 antibodies (h#32A1-T1, h#32A1-T2, h#32A1-T3, h#32A1-T4, h#32A1-T5, h#32A1-T6 , h#32A1-H1-1/L5, h#32A1-H5/L5, h#32A1-H1-1/L2-16 and h#32A1-H1-1/L2-15), it was observed that Potent activity in inhibiting osteoclast formation substantially comparable to that of rat anti-mouse Siglec-15 monoclonal antibody (#32A1 antibody) or human chimeric antibody of #32A1 antibody.

Example 30

Effect of humanized antibody supplemented with rat anti-mouse Siglec-15 monoclonal antibody #32A1 on the bone resorption activity of normal human osteoclast precursor cells (evaluated using collagen-coated plates)

Normal human osteoclast precursor cells (normal human natural osteoclast precursor cells, purchased from Sanko ^Junyaku Co., Ltd., cat. no. 2T-110) were seeded in 96-well OsteoLyse cell culture plates (purchased from Sanko Junyaku Co., Ltd., catalog number PA-1500). Minimal Essential Medium for Osteoclast Precursor Cells (OPBM, purchased from Sanko Junyaku Co., Ltd., cat. No. PT-8201) as the culture medium, the OPGM Supplement Set contains fetal bovine serum (final concentration: 10%), human RANKL (final concentration: 63.8 ng/ml), human M-CSF (final concentration: 33 ng/ml) Wait. To the obtained culture supernatant, each of the rat anti-mouse Siglec-15 monoclonal antibodies (#32A1 antibody) prepared in Example 6 was added at a final concentration of 4, 20, 100, or 500 ng/ml , the human chimeric antibody of the #32A1 antibody prepared in Example 19, and the 10 types of humanized #32A1 antibodies prepared in Examples 24 and 28 (h#32A1-T1, h#32A1-T2, h# 32A1-T3, h#32A1-T4, h#32A1-T5, h#32A1-T6, h#32A1-H1-1/L5, h#32A1-H5/L5, h#32A1-H1-1/L2- 16 and h#32A1-H1-1/L2-15), culture the cells in a CO ₂ incubator for 5 days. A 10 µl aliquot of the culture supernatant was collected, and 200 µl of the fluorophore-releasing reagent included in the OsteoLyse assay kit was added thereto, using a fluorescent plate reader (ARVO MX, manufactured by Perkin Elmer Inc.), and subjected to appropriate use. Time-resolved fluorometry (time-resolved fluorescence fluorometer) of the fluorescence measurement of europium (excitation: 340 nm, emission: 615 nm), whereby the amount of free fluorescent collagen fragments released in the culture supernatant was determined (Figures 48 and 49 ). As a result, addition of rat #32A1 antibody or human chimeric #32A1 antibody reduced the amount of fluorescent collagen fragments increased by addition of RANKL in a concentration-dependent manner. In the same manner, concentration-dependent inhibition by all 10 classes of humanized #32A1 antibodies examined was also observed. This result indicated that the bone resorption activity of human osteoclasts was inhibited by the humanized #32A1 antibody specifically binding to Siglec-15 protein.

Example 31

Biological Evaluation of Humanized Rat Anti-Mouse Siglec-15 mAb #32A1 Using Ovariectomized Rats

By the method described in Example 15, the inhibitory effect on the decrease in bone mineral density in ovariectomized rats exhibited by the humanized antibodies obtained in Examples 24 and 28 could be evaluated.

Example 32

Biological activity of humanized rat anti-mouse Siglec-15 monoclonal antibody #32A1 was evaluated based on TNFα-stimulated mouse osteoclastogenesis assay

By the method described in Example 26, the inhibitory effect of the humanized antibodies obtained in Examples 24 and 28 on osteoclastogenesis in mice stimulated by TNFα could be evaluated.

Example 33

Determination of Thermal Stability of Humanized Antibodies Using Differential Scanning Calorimetry (DSC)

Thermal stability was determined for h#32A1-H5/L5, h#32A1-H1-1/L5, h#32A1-H1-1/L2-15 and h#32A1-H1-1/L2-16. Each of these samples was prepared at a concentration of 0.5 mg/ml (in CBS solution), and a 400 μl aliquot thereof was used as a sample solution for DSC measurement. Conditions for the DSC measurement were set as follows. That is, the initial temperature is set to 20ºC, the final temperature is set to 100ºC, the temperature increase rate is set to 60ºC/hour, and the filtration time is set to 10 seconds. CBS was used as a reference solution. A VP-capillary DSC platform produced by MicroCal Inc. USA was used as an instrument for performing DSC measurements. Baseline correction was performed by subtracting the baseline (the scan curve obtained by also filling the sample cell with the reference solution) from the scan curve obtained for each sample solution. Figure 50 shows the thermogram of h#32A1-H5/L5, Figure 51 shows the thermogram of h#32A1-H1-1/L5, Figure 52 shows the thermogram of h#32A1-H1-1/L2-15 Thermogram, and Figure 53 shows the thermogram of h#32A1-H1-1/L2-16. When the peak top value in each thermogram is taken as the thermal denaturation midpoint temperature (Tm) in the Fab region, the Tm value of h#32A1-H5/L5 is 81.2ºC, h#32A1-H1-1/ The Tm value of L5 is 84.1ºC, the Tm value of h#32A1-H1-1/L2-15 is 80.0ºC, and the Tm value of h#32A1-H1-1/L2-16 (IgG2) is 77.9ºC.

Industrial Applicability

The chimeric or humanized anti-Siglec-15 antibody of the present invention has the ability to inhibit osteoclast differentiation or bone resorption activity, and the pharmaceutical composition containing the anti-Siglec-15 antibody can be a disease of abnormal bone metabolism therapeutic or preventive agents.

Accession number FERM BP-10999.

sequence listing

<110> DAIICHI SANKYO COMPANY, LIMITED

<120> anti-Siglec-15 antibody

<130> DSPCT-FP1021

<150> JP 2009-94613

<151> 2009-04-09

<160> 106

<170> PatentIn version 3.4

<210> 1

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<213> Homo sapiens

<220>

<221> CDS

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<223> Inventors: Hiruma, Yoshiharu; Tsuda, Eisuke

<223> Inventors: Takizawa, Takeshi; Nakayama, Makiko

<400> 1

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Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

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Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

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Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

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Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

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Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

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Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

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gcg gcg cgg ggc agc gag ctc tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

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cgc ttc cgg ctg ctg ggc aac ccg cgc cgc aac gac ctc tcg ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

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gtc gag cgc ctc gcc ctg gct gac gac cgc cgc tac ttc tgc cgc gtc 432

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

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Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

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Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

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Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

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Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

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Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

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Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

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Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

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Gly Ala Ser Gly Ala Ser Thr Val Ala Leu Leu Leu Gly Ala Leu Gly

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Phe Lys Ala Leu Leu Leu Leu Gly Val Leu Ala Ala Arg Ala Ala Arg

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Arg Arg Pro Glu His Leu Asp Thr Pro Asp Thr Pro Pro Arg Ser Gln

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Ala Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Asn Pro Arg Ser

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Pro Pro Ala Thr Met Cys Ser Pro

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Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

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Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

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Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

Gly Ala Ser Gly Ala Ser Thr Val Ala Leu Leu Leu Gly Ala Leu Gly

260 265 270

Phe Lys Ala Leu Leu Leu Leu Gly Val Leu Ala Ala Arg Ala Ala Arg

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Arg Arg Pro Glu His Leu Asp Thr Pro Asp Thr Pro Pro Arg Ser Gln

290 295 300

Ala Gln Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Asn Pro Arg Ser

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Pro Pro Ala Thr Met Cys Ser Pro

325

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Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

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Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

aac aca gag gcg cac agt gcc ccg gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

ccc gcg gag gtg aac gcg gag gct ggc gac gcg gcg gtg ctg ccc tgc 192

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

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Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

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Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

gcg gcg ccg ggc agc gag ctg tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgc ctg ctg ggc aac ccg cgc cgc aac gac ctg tcc ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gcg gac agc ggc cgc tac ttc tgc cgc gtg 432

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc acc ggc gac gcc cac gat cgc tat gag agt cgc cat ggg gtc 480

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgt ctg cgc gtg act gct gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

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ccg ggc ccc gcg cac gcc ttc cgc gcg ctc tgc acc gcc gag ggg gag 576

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccc ccg ccc gcc ctc gcc tgg tcg ggt ccc gcc cca ggc aac agc tcc 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

gct gcc ctg cag ggc cag ggt cac ggc tac cag gtg acc gcc gag ttg 672

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

ccc gcg ctg acc cgc gac ggc cgc tac acg tgc acg gcg gcc aat agc 720

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

ctg ggc cgc gcc gag gcc agc gtc tac ctg ttc cgc ttc cac ggc gcc 768

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

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Pro Gly Thr Ser Thr Leu Ala Leu Leu Leu Gly Ala Leu Gly Leu Lys

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Ala Leu Leu Leu Leu Leu Gly Ile Leu Gly Ala Arg Ala Thr Arg Arg Arg

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Leu Asp His Leu Val Pro Gln Asp Thr Pro Pro Arg Ser Gln Ala Gln

290 295 300

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Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Ser Pro Pro Gly His Gln

305 310 315 320

ctg cca cgt gtt tgc tgt gag gaa ctc ctc agc cat cac cat cta gtc 1008

Leu Pro Arg Val Cys Cys Glu Glu Leu Leu Ser His His His Leu Val

325 330 335

att cac cat gag aaa taa 1026

Ile His His Glu Lys

340

<210> 4

<211> 341

<212> PRT

<213> Mus musculus

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Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

Pro Gly Thr Ser Thr Leu Ala Leu Leu Leu Gly Ala Leu Gly Leu Lys

260 265 270

Ala Leu Leu Leu Leu Leu Gly Ile Leu Gly Ala Arg Ala Thr Arg Arg Arg

275 280 285

Leu Asp His Leu Val Pro Gln Asp Thr Pro Pro Arg Ser Gln Ala Gln

290 295 300

Glu Ser Asn Tyr Glu Asn Leu Ser Gln Met Ser Pro Pro Gly His Gln

305 310 315 320

Leu Pro Arg Val Cys Cys Glu Glu Leu Leu Ser His His His Leu Val

325 330 335

Ile His His Glu Lys

340

<210> 5

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<221> CDS

<222> (1)..(774)

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Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

atg gga tcc ctt gtg aaa act aga aga gac gct tcg ggg gat ctg ctc 96

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

aac aca gag gcg cac agt gcc ccg gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

ccc gcg gag gtg aac gcg gag gct ggc gac gcg gcg gtg ctg ccc tgc 192

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc tcg ggc gag ccg tac gcg ggc ccg cag gtg ttc cgc tgc acc 288

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

gcg gcg ccg ggc agc gag ctg tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgc ctg ctg ggc aac ccg cgc cgc aac gac ctg tcc ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gcg gac agc ggc cgc tac ttc tgc cgc gtg 432

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc acc ggc gac gcc cac gat cgc tat gag agt cgc cat ggg gtc 480

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgt ctg cgc gtg act gct gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccg ggc ccc gcg cac gcc ttc cgc gcg ctc tgc acc gcc gag ggg gag 576

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccc ccg ccc gcc ctc gcc tgg tcg ggt ccc gcc cca ggc aac agc tcc 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

gct gcc ctg cag ggc cag ggt cac ggc tac cag gtg acc gcc gag ttg 672

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

ccc gcg ctg acc cgc gac ggc cgc tac acg tgc acg gcg gcc aat agc 720

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

ctg ggc cgc gcc gag gcc agc gtc tac ctg ttc cgc ttc cac ggc gcc 768

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

ccc gga 774

Pro Gly

<210> 6

<211> 258

<212> PRT

<213> Mus musculus

<400> 6

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

Pro Gly

<210> 7

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for soluble mouse Siglec-15 cDNA

<400> 7

ggggacaagt ttgtacaaaa aagcaggctt caccatggag gggtccctcc aactc 55

<210> 8

<211> 55

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for soluble mouse Siglec-15 cDNA

<400> 8

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<210> 9

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Sequencing primers for soluble mouse Siglec-15 cDNA

<400> 9

tgcgtgaagg tgcagggcag 20

<210> 10

<211> 17

<212>DNA

<213> Artificial sequence

<220>

<223> Sequencing primers for soluble mouse Siglec-15 cDNA

<400> 10

cctcgcctgg tcgggtc 17

<210> 11

<211> 891

<212>DNA

<213> Artificial sequence

<220>

<223> His-tagged mouse Siglec-15 cDNA

<220>

<221> CDS

<222> (1)..(891)

<400> 11

atg gag ggg tcc ctc caa ctc ctg gcc tgc ttg gcc tgt gtg ctc cag 48

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

atg gga tcc ctt gtg aaa act aga aga gac gct tcg ggg gat ctg ctc 96

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

aac aca gag gcg cac agt gcc ccg gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

ccc gcg gag gtg aac gcg gag gct ggc gac gcg gcg gtg ctg ccc tgc 192

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc tcg ggc gag ccg tac gcg ggc ccg cag gtg ttc cgc tgc acc 288

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

gcg gcg ccg ggc agc gag ctg tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgc ctg ctg ggc aac ccg cgc cgc aac gac ctg tcc ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gcg gac agc ggc cgc tac ttc tgc cgc gtg 432

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc acc ggc gac gcc cac gat cgc tat gag agt cgc cat ggg gtc 480

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgt ctg cgc gtg act gct gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccg ggc ccc gcg cac gcc ttc cgc gcg ctc tgc acc gcc gag ggg gag 576

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccc ccg ccc gcc ctc gcc tgg tcg ggt ccc gcc cca ggc aac agc tcc 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

gct gcc ctg cag ggc cag ggt cac ggc tac cag gtg acc gcc gag ttg 672

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

ccc gcg ctg acc cgc gac ggc cgc tac acg tgc acg gcg gcc aat agc 720

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

ctg ggc cgc gcc gag gcc agc gtc tac ctg ttc cgc ttc cac ggc gcc 768

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

ccc gga gac cca gct ttc ttg tac aaa gtg gtt gat atc gaa ggt cgt 816

Pro Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Glu Gly Arg

260 265 270

ggg ggt aag cct atc cct aac cct ctc ctc ggt ctc gat tct acg cgt 864

Gly Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg

275 280 285

acc ggt cat cat cac cat cac cat tga 891

Thr Gly His His His His His His His His

290 295

<210> 12

<211> 296

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 12

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

Pro Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Glu Gly Arg

260 265 270

Gly Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg

275 280 285

Thr Gly His His His His His His His His

290 295

<210> 13

<211> 1512

<212>DNA

<213> Artificial sequence

<220>

<223> Fc-fused mouse Siglec-15 cDNA

<220>

<221> CDS

<222> (1)..(1512)

<400> 13

atg gag ggg tcc ctc caa ctc ctg gcc tgc ttg gcc tgt gtg ctc cag 48

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

atg gga tcc ctt gtg aaa act aga aga gac gct tcg ggg gat ctg ctc 96

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

aac aca gag gcg cac agt gcc ccg gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

ccc gcg gag gtg aac gcg gag gct ggc gac gcg gcg gtg ctg ccc tgc 192

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc tcg ggc gag ccg tac gcg ggc ccg cag gtg ttc cgc tgc acc 288

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

gcg gcg ccg ggc agc gag ctg tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgc ctg ctg ggc aac ccg cgc cgc aac gac ctg tcc ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gcg gac agc ggc cgc tac ttc tgc cgc gtg 432

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc acc ggc gac gcc cac gat cgc tat gag agt cgc cat ggg gtc 480

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgt ctg cgc gtg act gct gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccg ggc ccc gcg cac gcc ttc cgc gcg ctc tgc acc gcc gag ggg gag 576

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccc ccg ccc gcc ctc gcc tgg tcg ggt ccc gcc cca ggc aac agc tcc 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

gct gcc ctg cag ggc cag ggt cac ggc tac cag gtg acc gcc gag ttg 672

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

ccc gcg ctg acc cgc gac ggc cgc tac acg tgc acg gcg gcc aat agc 720

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

ctg ggc cgc gcc gag gcc agc gtc tac ctg ttc cgc ttc cac ggc gcc 768

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

ccc gga gac cca gct ttc ttg tac aaa gtg gtt gat atc cag gca gag 816

Pro Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Gln Ala Glu

260 265 270

ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct 864

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag 912

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 960

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac 1008

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac 1056

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 1104

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc 1152

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

370 375 380

cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1200

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag 1248

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

405 410 415

aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 1296

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1344

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc 1392

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca 1440

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc 1488

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

ctc tcc ctg tct ccg ggt aaa taa 1512

Leu Ser Leu Ser Pro Gly Lys

500

<210> 14

<211> 503

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 14

Met Glu Gly Ser Leu Gln Leu Leu Ala Cys Leu Ala Cys Val Leu Gln

1 5 10 15

Met Gly Ser Leu Val Lys Thr Arg Arg Asp Ala Ser Gly Asp Leu Leu

20 25 30

Asn Thr Glu Ala His Ser Ala Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Ala Glu Val Asn Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ser Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Thr

85 90 95

Ala Ala Pro Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Ser Gly Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Thr Gly Asp Ala His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu Arg Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Gly Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Pro Gly Asn Ser Ser

195 200 205

Ala Ala Leu Gln Gly Gln Gly His Gly Tyr Gln Val Thr Ala Glu Leu

210 215 220

Pro Ala Leu Thr Arg Asp Gly Arg Tyr Thr Cys Thr Ala Ala Asn Ser

225 230 235 240

Leu Gly Arg Ala Glu Ala Ser Val Tyr Leu Phe Arg Phe His Gly Ala

245 250 255

Pro Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Gln Ala Glu

260 265 270

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

275 280 285

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

290 295 300

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

305 310 315 320

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

325 330 335

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

340 345 350

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

355 360 365

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

370 375 380

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

385 390 395 400

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

405 410 415

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

420 425 430

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

435 440 445

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

450 455 460

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

465 470 475 480

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

485 490 495

Leu Ser Leu Ser Pro Gly Lys

500

<210> 15

<211> 780

<212>DNA

<213> Homo sapiens

<220>

<221> CDRs

<222> (1)..(780)

<220>

<221> CDS

<222> (1)..(780)

<400> 15

atg gaa aag tcc atc tgg ctg ctg gcc tgc ttg gcg tgg gtt ctc ccg 48

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

aca ggc tca ttt gtg aga act aaa ata gat act acg gag aac ttg ctc 96

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

aac aca gag gtg cac agc tcg cca gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

cca ccc gag gtg agc gcg gag gca ggc gac gcg gca gtg ctg ccc tgc 192

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc gcg ggc gag ccc tat gcg ggc ccg cag gtg ttc cgc tgc gct 288

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

gcg gcg cgg ggc agc gag ctc tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgg ctg ctg ggc aac ccg cgc cgc aac gac ctc tcg ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gct gac gac cgc cgc tac ttc tgc cgc gtc 432

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc gcc ggc gac gtc cat gac cgc tac gag agc cgc cac ggc gtc 480

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgg ctg cac gtg aca gcc gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccc agt ccg gct cac gcc ttc cgc gcg ctc tgc act gcc gaa ggg gag 576

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccg ccg ccc gcc ctc gcc tgg tcc ggc ccg gcc ctg ggc aac agc ttg 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

gca gcc gtg cgg agc ccg cgt gag ggt cac ggc cac cta gtg acc gcc 672

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

gaa ctg ccc gca ctg acc cat gac ggc cgc tac acg tgt acg gcc gcc 720

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

aac agc ctg ggc cgc tcc gag gcc agc gtc tac ctg ttc cgc ttc cat 768

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

ggc gcc agc ggg 780

Gly Ala Ser Gly

260

<210> 16

<211> 260

<212> PRT

<213> Homo sapiens

<400> 16

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

Gly Ala Ser Gly

260

<210> 17

<211> 56

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for soluble human Siglec-15 cDNA

<400> 17

ggggacaagt ttgtacaaaa aagcaggctt caccatggaa aagtccatct ggctgc 56

<210> 18

<211> 52

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for soluble human Siglec-15 cDNA

<400> 18

ggggaccact ttgtacaaga aagctgggtc cccgctggcg ccatggaagc gg 52

<210> 19

<211> 897

<212>DNA

<213> Artificial sequence

<220>

<223> His-tagged human Siglec-15 cDNA

<220>

<221> CDS

<222> (1)..(897)

<400> 19

atg gaa aag tcc atc tgg ctg ctg gcc tgc ttg gcg tgg gtt ctc ccg 48

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

aca ggc tca ttt gtg aga act aaa ata gat act acg gag aac ttg ctc 96

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

aac aca gag gtg cac agc tcg cca gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

cca ccc gag gtg agc gcg gag gca ggc gac gcg gca gtg ctg ccc tgc 192

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc gcg ggc gag ccc tat gcg ggc ccg cag gtg ttc cgc tgc gct 288

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

gcg gcg cgg ggc agc gag ctc tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgg ctg ctg ggc aac ccg cgc cgc aac gac ctc tcg ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gct gac gac cgc cgc tac ttc tgc cgc gtc 432

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc gcc ggc gac gtc cat gac cgc tac gag agc cgc cac ggc gtc 480

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgg ctg cac gtg aca gcc gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccc agt ccg gct cac gcc ttc cgc gcg ctc tgc act gcc gaa ggg gag 576

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccg ccg ccc gcc ctc gcc tgg tcc ggc ccg gcc ctg ggc aac agc ttg 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

gca gcc gtg cgg agc ccg cgt gag ggt cac ggc cac cta gtg acc gcc 672

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

gaa ctg ccc gca ctg acc cat gac ggc cgc tac acg tgt acg gcc gcc 720

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

aac agc ctg ggc cgc tcc gag gcc agc gtc tac ctg ttc cgc ttc cat 768

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

ggc gcc agc ggg gac cca gct ttc ttg tac aaa gtg gtt gat atc gaa 816

Gly Ala Ser Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Glu

260 265 270

ggt cgt ggg ggt aag cct atc cct aac cct ctc ctc ggt ctc gat tct 864

Gly Arg Gly Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser

275 280 285

acg cgt acc ggt cat cat cac cat cac cat tga 897

Thr Arg Thr Gly His His His His His His His His

290 295

<210> 20

<211> 298

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 20

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

Gly Ala Ser Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Glu

260 265 270

Gly Arg Gly Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser

275 280 285

Thr Arg Thr Gly His His His His His His His His

290 295

<210> 21

<211> 1518

<212>DNA

<213> Artificial sequence

<220>

<223> Fc-fused human Siglec-15 cDNA

<220>

<221> CDS

<222> (1)..(1518)

<400> 21

atg gaa aag tcc atc tgg ctg ctg gcc tgc ttg gcg tgg gtt ctc ccg 48

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

aca ggc tca ttt gtg aga act aaa ata gat act acg gag aac ttg ctc 96

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

aac aca gag gtg cac agc tcg cca gcg cag cgc tgg tcc atg cag gtg 144

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

cca ccc gag gtg agc gcg gag gca ggc gac gcg gca gtg ctg ccc tgc 192

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

acc ttc acg cac ccg cac cgc cac tac gac ggg ccg ctg acg gcc atc 240

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

tgg cgc gcg ggc gag ccc tat gcg ggc ccg cag gtg ttc cgc tgc gct 288

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

gcg gcg cgg ggc agc gag ctc tgc cag acg gcg ctg agc ctg cac ggc 336

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

cgc ttc cgg ctg ctg ggc aac ccg cgc cgc aac gac ctc tcg ctg cgc 384

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

gtc gag cgc ctc gcc ctg gct gac gac cgc cgc tac ttc tgc cgc gtc 432

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

gag ttc gcc ggc gac gtc cat gac cgc tac gag agc cgc cac ggc gtc 480

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

cgg ctg cac gtg aca gcc gcg ccg cgg atc gtc aac atc tcg gtg ctg 528

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

ccc agt ccg gct cac gcc ttc cgc gcg ctc tgc act gcc gaa ggg gag 576

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

ccg ccg ccc gcc ctc gcc tgg tcc ggc ccg gcc ctg ggc aac agc ttg 624

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

gca gcc gtg cgg agc ccg cgt gag ggt cac ggc cac cta gtg acc gcc 672

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

gaa ctg ccc gca ctg acc cat gac ggc cgc tac acg tgt acg gcc gcc 720

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

aac agc ctg ggc cgc tcc gag gcc agc gtc tac ctg ttc cgc ttc cat 768

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

ggc gcc agc ggg gac cca gct ttc ttg tac aaa gtg gtt gat atc cag 816

Gly Ala Ser Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Gln

260 265 270

gca gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca 864

Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

275 280 285

gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa 912

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

290 295 300

ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg 960

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

305 310 315 320

gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac 1008

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

325 330 335

gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag 1056

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

340 345 350

cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac 1104

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

355 360 365

cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa 1152

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

370 375 380

gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag 1200

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

385 390 395 400

ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg 1248

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

405 410 415

acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc 1296

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

420 425 430

agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac 1344

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

435 440 445

tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc 1392

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

450 455 460

tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc 1440

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

465 470 475 480

ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag 1488

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

485 490 495

aag agc ctc tcc ctg tct ccg ggt aaa taa 1518

Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505

<210> 22

<211> 505

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 22

Met Glu Lys Ser Ile Trp Leu Leu Ala Cys Leu Ala Trp Val Leu Pro

1 5 10 15

Thr Gly Ser Phe Val Arg Thr Lys Ile Asp Thr Thr Glu Asn Leu Leu

20 25 30

Asn Thr Glu Val His Ser Ser Pro Ala Gln Arg Trp Ser Met Gln Val

35 40 45

Pro Pro Glu Val Ser Ala Glu Ala Gly Asp Ala Ala Val Leu Pro Cys

50 55 60

Thr Phe Thr His Pro His Arg His Tyr Asp Gly Pro Leu Thr Ala Ile

65 70 75 80

Trp Arg Ala Gly Glu Pro Tyr Ala Gly Pro Gln Val Phe Arg Cys Ala

85 90 95

Ala Ala Arg Gly Ser Glu Leu Cys Gln Thr Ala Leu Ser Leu His Gly

100 105 110

Arg Phe Arg Leu Leu Gly Asn Pro Arg Arg Asn Asp Leu Ser Leu Arg

115 120 125

Val Glu Arg Leu Ala Leu Ala Asp Asp Arg Arg Tyr Phe Cys Arg Val

130 135 140

Glu Phe Ala Gly Asp Val His Asp Arg Tyr Glu Ser Arg His Gly Val

145 150 155 160

Arg Leu His Val Thr Ala Ala Pro Arg Ile Val Asn Ile Ser Val Leu

165 170 175

Pro Ser Pro Ala His Ala Phe Arg Ala Leu Cys Thr Ala Glu Gly Glu

180 185 190

Pro Pro Pro Ala Leu Ala Trp Ser Gly Pro Ala Leu Gly Asn Ser Leu

195 200 205

Ala Ala Val Arg Ser Pro Arg Glu Gly His Gly His Leu Val Thr Ala

210 215 220

Glu Leu Pro Ala Leu Thr His Asp Gly Arg Tyr Thr Cys Thr Ala Ala

225 230 235 240

Asn Ser Leu Gly Arg Ser Glu Ala Ser Val Tyr Leu Phe Arg Phe His

245 250 255

Gly Ala Ser Gly Asp Pro Ala Phe Leu Tyr Lys Val Val Asp Ile Gln

260 265 270

Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

275 280 285

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

290 295 300

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

305 310 315 320

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

325 330 335

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

340 345 350

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

355 360 365

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

370 375 380

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

385 390 395 400

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

405 410 415

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

420 425 430

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

435 440 445

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

450 455 460

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

465 470 475 480

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

485 490 495

Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505

<210> 23

<211> 33

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for heavy chain cDNA of rat #32A1 antibody

<400> 23

ggccgggtgg gctacgttgc aggtgacggt ctg 33

<210> 24

<211> 35

<212>DNA

<213> Artificial sequence

<220>

<223> PCR Primers for Light Chain cDNA of Rat #32A1 Antibody

<400> 24

catgctgtac gtgctgtctt tgctgtcctg atcag 35

<210> 25

<211> 29

<212>DNA

<213> Artificial sequence

<220>

<223> Sequencing primers for heavy chain cDNA of rat #32A1 antibody

<400> 25

ctccagagtt ccaggtcacg gtgactggc 29

<210> 26

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Sequenced promoter of light chain cDNA of rat #32A1 antibody

<400> 26

tccagttgct aactgttccg 20

<210> 27

<211> 501

<212>DNA

<213> Rattus norvegicus

<220>

<221> CDA

<222> (1)..(501)

<220>

<221> CDS

<222> (1)..(501)

<400> 27

atg aag ttg tgg ttg agc tgg ata ttc ctt gtt gtt ctt ttc aaa ggt 48

Met Lys Leu Trp Leu Ser Trp Ile Phe Leu Val Val Leu Phe Lys Gly

1 5 10 15

gtg agg tgt gag gtg caa att ttg gag act gga gga ggc ttg gtg aag 96

Val Arg Cys Glu Val Gln Ile Leu Glu Thr Gly Gly Gly Leu Val Lys

20 25 30

ccc ggt ggt tcc ctg aga ctg tct tgt gca acg tct gga ttc aat ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aat gat tat ttc atg aac tgg gtc cgt cag gct cca gaa aag ggg cta 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

gag tgg gtt gct caa ata agg aac aaa att tat act tat gcc aca ttt 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tat gcg gag tct ttg gaa ggc aga gtc aca atc tca cga gac gat tcc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

gaa agc agt gtc tac ctg caa gtg agc agt tta aga gct gaa gac act 336

Glu Ser Ser Val Tyr Leu Gln Val Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc att tat tac tgt aca aga tcc cta act ggg gga gac tac ttt gat 384

Ala Ile Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc caa gga gtc atg gtc aca gtc tcc tta gct gaa aca aca 432

Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Leu Ala Glu Thr Thr

130 135 140

gcc cca tct gtc tat cca ctg gct cct gga act gct ctc aaa agt aac 480

Ala Pro Ser Val Tyr Pro Leu Ala Pro Gly Thr Ala Leu Lys Ser Asn

145 150 155 160

tcc atg gtg acc ctg gga tgc 501

Ser Met Val Thr Leu Gly Cys

165

<210> 28

<211> 167

<212> PRT

<213> Rattus norvegicus

<400> 28

Met Lys Leu Trp Leu Ser Trp Ile Phe Leu Val Val Leu Phe Lys Gly

1 5 10 15

Val Arg Cys Glu Val Gln Ile Leu Glu Thr Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

Glu Ser Ser Val Tyr Leu Gln Val Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Ile Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Leu Ala Glu Thr Thr

130 135 140

Ala Pro Ser Val Tyr Pro Leu Ala Pro Gly Thr Ala Leu Lys Ser Asn

145 150 155 160

Ser Met Val Thr Leu Gly Cys

165

<210> 29

<211> 417

<212>DNA

<213> Rattus norvegicus

<220>

<221> CDS

<222> (1)..(417)

<400> 29

atg gag aca gac aga ctc ctg cta tgg gca ctg ctg ctc tgg gtt cca 48

Met Glu Thr Asp Arg Leu Leu Leu Trp Ala Leu Leu Leu Trp Val Pro

1 5 10 15

ggc tcc act ggt gac att gtc ttg acc cag tct cct gct ttg gct gtg 96

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

tct cta ggg cag agg gcc aca atc tcc tgt agg gcc agc caa agt gtc 144

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

act att tct gga tat agt ttt ata cac tgg tac caa cag aaa cca gga 192

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

cag caa ccc aga ctc ctc atc tat cgt gca tcc aac cta gcc tct ggg 240

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

atc ccg gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc acc ctc 288

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

acc atc aat cct gtg cag gct gat gat att gca acc tat ttc tgt cag 336

Thr Ile Asn Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Phe Cys Gln

100 105 110

cag agt agg aaa tct ccg tgg acg ttc gct gga ggc acc aag ctg gaa 384

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

ttg aga cgg gct gat gct gca cca act gta tct 417

Leu Arg Arg Ala Asp Ala Ala Pro Thr Val Ser

130 135

<210> 30

<211> 139

<212> PRT

<213> Rattus norvegicus

<400> 30

Met Glu Thr Asp Arg Leu Leu Leu Trp Ala Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Asn Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Phe Cys Gln

100 105 110

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

Leu Arg Arg Ala Asp Ala Ala Pro Thr Val Ser

130 135

<210> 31

<211> 28

<212>DNA

<213> Artificial sequence

<220>

<223> Primer EFF1

<400> 31

ccacgcgccc tgtagcggcg cattaagc 28

<210> 32

<211> 30

<212>DNA

<213> Artificial sequence

<220>

<223> Primer EFsmaR

<400> 32

aaacccggga gctttttgca aaagcctagg 30

<210> 33

<211> 1704

<212>DNA

<213> Artificial sequence

<220>

<223> Fragment B

<400> 33

ggtaccaccc aagctggcta ggtaagcttg ctagcgccac catggtgctg cagacccagg 60

tgttcatctc cctgctgctg tggatctccg gcgcatatgg cgatatcgtg atgattaaac 120

gtacggtggc cgccccctcc gtgttcatct tccccccctc cgacgagcag ctgaagtccg 180

gcaccgcctc cgtggtgtgc ctgctgaata acttctaccc cagagaggcc aaggtgcagt 240

ggaaggtgga caacgccctg cagtccggga actcccagga gagcgtgacc gagcaggaca 300

gcaaggacag cacctacagc ctgagcagca ccctgaccct gagcaaagcc gactacgaga 360

agcacaaggt gtacgcctgc gaggtgaccc accagggcct gagctccccc gtcaccaaga 420

gcttcaacag ggggggagtgt tagggccccg tttaaacggg tggcatccct gtgaccccctc 480

cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 540

aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 600

gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 660

ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 720

ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac 780

caggctcacc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 840

ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 900

acaggcgtga accactgctc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 960

tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 1020

tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 1080

tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 1140

gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 1200

agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 1260

cggtctattc ttttgatta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 1320

agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg 1380

tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc 1440

agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca 1500

tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc 1560

gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 1620

cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 1680

aggcttttgc aaaaagctcc cggg 1704

<210> 34

<211> 1120

<212>DNA

<213> Artificial sequence

<220>

<223> Human IgG1 signal + human IgG1 constant region

<400> 34

tgctagcgcc accatgaaac acctgtggtt cttcctcctg ctggtggcag ctcccagatg 60

ggtgctgagc caggtgcaat tgtgcaggcg gttagctcag cctccaccaa gggcccaagc 120

gtcttccccc tggcaccctc ctccaagagc acctctggcg gcacagccgc cctgggctgc 180

ctggtcaagg actacttccc cgaacccgtg accgtgagct ggaactcagg cgccctgacc 240

agcggcgtgc acaccttccc cgctgtcctg cagtcctcag gactctactc cctcagcagc 300

gtggtgaccg tgccctccag cagcttgggc accccagacct acatctgcaa cgtgaatcac 360

aagcccagca acaccaaggt ggacaagaga gttgagccca aatcttgtga caaaactcac 420

acatgcccac cctgcccagc acctgaactc ctggggggac cctcagtctt cctcttcccc 480

ccaaaaccca aggacacccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 540

gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 600

cataatgcca agacaaagcc ccgggaggag cagtacaaca gcacgtaccg ggtggtcagc 660

gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 720

aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg ccagccccgg 780

gaaccacagg tgtacacccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 840

ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 900

ggccagcccg agaacaacta caagaccacc cctcccgtgc tggactccga cggctccttc 960

ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcagggcaa cgtcttctca 1020

tgctccgtga tgcatgaggc tctgcacaac cactacaccc agaagagcct ctccctgtct 1080

cccggcaaat gagatatcgg gcccgtttaa acgggtggca 1120

<210> 35

<211> 40

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for heavy chain variable region cDNA of #32A1 antibody

<400> 35

aaagctgagc gaggtgcaaa ttttggagac tggaggaggc 40

<210> 36

<211> 39

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for heavy chain variable region cDNA of #32A1 antibody

<400> 36

aaagctgagc tgactgtgac catgactcct tggccccag 39

<210> 37

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Sequencing primers for heavy chain variable region cDNA of #32A1 antibody

<400> 37

taatacgact cactataggg 20

<210> 38

<211> 42

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for light chain variable region cDNA of #32A1 antibody

<400> 38

aaacatatgg cgacattgtc ttgacccagt ctcctgcttt gg 42

<210> 39

<211> 37

<212>DNA

<213> Artificial sequence

<220>

<223> PCR primers for light chain variable region cDNA of #32A1 antibody

<400> 39

aaacgtacgt ctcaattcca gcttggtgcc tccagcg 37

<210> 40

<211> 1413

<212>DNA

<213> Artificial sequence

<220>

<223> Chimeric heavy chain of #32A1

<220>

<221> CDS

<222> (1)..(1413)

<400> 40

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg caa att ttg gag act gga gga ggc ttg gtg aag 96

Val Leu Ser Glu Val Gln Ile Leu Glu Thr Gly Gly Gly Leu Val Lys

20 25 30

ccc ggt ggt tcc ctg aga ctg tct tgt gca acg tct gga ttc aat ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aat gat tat ttc atg aac tgg gtc cgt cag gct cca gaa aag ggg cta 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

gag tgg gtt gct caa ata agg aac aaa att tat act tat gcc aca ttt 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tat gcg gag tct ttg gaa ggc aga gtc aca atc tca cga gac gat tcc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

gaa agc agt gtc tac ctg caa gtg agc agt tta aga gct gaa gac act 336

Glu Ser Ser Val Tyr Leu Gln Val Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc att tat tac tgt aca aga tcc cta act ggg gga gac tac ttt gat 384

Ala Ile Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc caa gga gtc atg gtc aca gtc agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa tga 1413

Ser Leu Ser Pro Gly Lys

465 470

<210> 41

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 41

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Ile Leu Glu Thr Gly Gly Gly Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

Glu Ser Ser Val Tyr Leu Gln Val Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Ile Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 42

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> Chimeric light chain of #32A1

<220>

<221> CDS

<222> (1)..(714)

<400> 42

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac att gtc ttg acc cag tct cct gct ttg gct gtg 96

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

tct cta ggg cag agg gcc aca atc tcc tgt agg gcc agc caa agt gtc 144

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

act att tct gga tat agt ttt ata cac tgg tac caa cag aaa cca gga 192

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

cag caa ccc aga ctc ctc atc tat cgt gca tcc aac cta gcc tct ggg 240

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

atc ccg gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc acc ctc 288

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

acc atc aat cct gtg cag gct gat gat att gca acc tat ttc tgt cag 336

Thr Ile Asn Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Phe Cys Gln

100 105 110

cag agt agg aaa tct ccg tgg acg ttc gct gga ggc acc aag ctg gaa 384

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

ttg aga cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc tcc 432

Leu Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg aat 480

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac gcc 528

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc aag 576

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc gac 624

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc ctg 672

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt tag 714

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 43

<211> 237

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 43

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Asn Pro Val Gln Ala Asp Asp Ile Ala Thr Tyr Phe Cys Gln

100 105 110

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

Leu Arg Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 44

<211> 5

<212> PRT

<213> Rattus norvegicus

<400> 44

Asp Tyr Phe Met Asn

1 5

<210> 45

<211> 19

<212> PRT

<213> Rattus norvegicus

<400> 45

Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu Ser

1 5 10 15

Leu Glu Gly

<210> 46

<211> 10

<212> PRT

<213> Rattus norvegicus

<400> 46

Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr

1 5 10

<210> 47

<211> 15

<212> PRT

<213> Rattus norvegicus

<400> 47

Arg Ala Ser Gln Ser Val Thr Ile Ser Gly Tyr Ser Phe Ile His

1 5 10 15

<210> 48

<211> 7

<212> PRT

<213> Rattus norvegicus

<400> 48

Arg Ala Ser Asn Leu Ala Ser

1 5

<210> 49

<211> 9

<212> PRT

<213> Rattus norvegicus

<400> 49

Gln Gln Ser Arg Lys Ser Pro Trp Thr

1 5

<210> 50

<211> 1410

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T1H

<220>

<221> CDS

<222> (1)..(1410)

<400> 50

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg caa ctg gtg gag agc ggc ggc gga ctt gtg caa 96

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctt agc tgt gcc gcc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga caa gcc cct ggc aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

gag tgg gtg gcc caa atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctt gag ggc aga ttc acc atc tcc aga gac aac gcc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

aag aac agc ctg tac ctt caa atg aac tcc ctg aga gcc gag gac acc 336

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt gcc aga agc ctt acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc caa ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa 1410

Ser Leu Ser Pro Gly Lys

465 470

<210> 51

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 51

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 52

<211> 1410

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T2H

<220>

<221> CDS

<222> (1)..(1410)

<400> 52

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg caa ctg gtg gag agc ggc ggc gga ctg gtg caa 96

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctg agc tgt gcc gcc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga caa gcc cct gag aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

gag tgg gtg gcc caa atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctg gag ggc aga gtg acc atc tcc aga gac aac gcc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ala

85 90 95

aag aac agc ctg tac ctg caa atg tcc tcc ctg aga gcc gag gac acc 336

Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt gcc aga agc ctg acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc caa ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa 1410

Ser Leu Ser Pro Gly Lys

465 470

<210> 53

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 53

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 54

<211> 1410

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T3H

<220>

<221> CDS

<222> (1)..(1410)

<400> 54

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg cag atc gtg gag agc ggc ggc gga ctt gtg cag 96

Val Leu Ser Glu Val Gln Ile Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctt agc tgt gcc acc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga cag gcc cct gag aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

gag tgg gtg gcc cag atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctt gag ggc aga gtg acc atc tcc aga gac gac agc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

gag tcc agc gtg tac ctt cag atg tcc agc ctg aga gcc gag gac acc 336

Glu Ser Ser Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt acc aga agc ctt acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc cag ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa 1410

Ser Leu Ser Pro Gly Lys

465 470

<210> 55

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 55

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Ile Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asp Ser

85 90 95

Glu Ser Ser Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 56

<211> 1410

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T5H

<220>

<221> CDS

<222> (1)..(1410)

<400> 56

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg cag gtg gtg gag agc ggc ggc gga ctt gtg cag 96

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctt agc tgt gcc acc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga cag gcc cct ggc aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

gag tgg gtg gcc cag atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctt gag ggc aga ttc acc atc tcc aga gac aac agc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

aag tcc acc gtg tac ctt cag atg aac tcc ctg aga gcc gag gac acc 336

Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt acc aga agc ctt acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc cag ggc acc ctg gtg acc gtg agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa 1410

Ser Leu Ser Pro Gly Lys

465 470

<210> 57

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 57

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 58

<211> 1410

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T6H

<220>

<221> CDS

<222> (1)..(1410)

<400> 58

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg cag gtg gtg gag agc ggc ggc gga ctt gtg cag 96

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctt agc tgt gcc acc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga cag gcc cct gag aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

gag tgg gtg gcc cag atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctt gag ggc aga gtg acc atc tcc aga gac aac agc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ser

85 90 95

aag tcc acc gtg tac ctt cag atg tcc agc ctt aga gcc gag gac acc 336

Lys Ser Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt acc aga agc ctt acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc cag ggc acc ctt gtg aca gtg agc tca gcc tcc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cca agc gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggc 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

ggc aca gcc gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccc 528

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc ccc gct gtc ctg cag tcc tca gga ctc tac tcc ctc agc agc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 672

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 720

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

aaa tct tgt gac aaa act cac aca tgc cca ccc tgc cca gca cct gaa 768

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

ctc ctg ggg gga ccc tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 816

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 864

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 912

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

gtg gag gtg cat aat gcc aag aca aag ccc cgg gag gag cag tac aac 960

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

agc acg tac cgg gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 1008

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1056

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggc cag ccc cgg gaa 1104

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 1152

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 1200

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

gcc gtg gag tgg gag agc aat ggc cag ccc gag aac aac tac aag acc 1248

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

acc cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1296

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

ctc acc gtg gac aag agc agg tgg cag cag ggc aac gtc ttc tca tgc 1344

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

tcc gtg atg cat gag gct ctg cac aac cac tac acc cag aag agc ctc 1392

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

tcc ctg tct ccc ggc aaa 1410

Ser Leu Ser Pro Gly Lys

465 470

<210> 59

<211> 470

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 59

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Ser Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

145 150 155 160

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

210 215 220

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro

225 230 235 240

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

245 250 255

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

260 265 270

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

275 280 285

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

290 295 300

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

305 310 315 320

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

325 330 335

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

340 345 350

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

355 360 365

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

370 375 380

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

405 410 415

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

420 425 430

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

435 440 445

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 60

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T1L

<220>

<221> CDS

<222> (1)..(714)

<400> 60

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg atg acc cag agc cct gac tcc ctt gcc 96

Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

gtg tcc ctg ggc gag aga gcc acc atc aac tgt aga gcc tcc cag agc 144

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

ggc cag cct cct aag ctt ctg atc tac aga gcc tcc aac ctt gcc agc 240

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc gtg cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc 288

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg acc atc agc tcc ctt cag gcc gag gac gtg gcc gtg tac tac tgt 336

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys

100 105 110

cag cag agc aga aag tcc cct tgg acc ttc ggc ggc ggc acc aag gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 61

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 61

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 62

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T2L

<220>

<221> CDS

<222> (1)..(714)

<400> 62

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg atg acc cag agc cct gac tcc ctt gcc 96

Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

gtg tcc ctg ggc gag aga gcc acc atc agc tgt aga gcc tcc cag agc 144

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

ggc cag cct cct aga ctt ctg atc tac aga gcc tcc aac ctt gcc agc 240

Gly Gln Pro Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc gtg cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc 288

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg acc atc agc tcc ctt cag gcc gag gac gtg gcc gtg tac ttc tgt 336

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys

100 105 110

cag cag agc aga aag tcc cct tgg acc ttc gcc ggc ggc acc aag gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 63

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 63

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 64

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T3L

<220>

<221> CDS

<222> (1)..(714)

<400> 64

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg ctt acc cag agc cct gac tcc ctt gcc 96

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

gtg tcc ctg ggc gag aga gcc acc atc agc tgt aga gcc tcc cag agc 144

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

ggc cag cag cct aga ctt ctg atc tac aga gcc tcc aac ctt gcc agc 240

Gly Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc atc cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc 288

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg acc atc agc tcc ctt cag gcc gag gac gtg gcc acc tac ttc tgt 336

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys

100 105 110

cag cag agc aga aag tcc cct tgg acc ttc gcc ggc ggc acc aag gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 65

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 65

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 66

<211> 711

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T4L

<220>

<221> CDS

<222> (1)..(711)

<400> 66

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg ctt acc cag agc cct gcc ctt gcc gtg 96

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

tcc ctg ggc gag aga gcc acc atc agc tgt aga gcc tcc cag agc gtg 144

Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct ggc 192

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

cag cag cct aga ctt ctg atc tac aga gcc tcc aac ctt gcc agc ggc 240

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

atc cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc ctg 288

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

acc atc agc tcc ctt cag gcc gag gac gtg gcc acc tac ttc tgt cag 336

Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys Gln

100 105 110

cag agc aga aag tcc cct tgg acc ttc gcc ggc ggc acc aag ctg gag 384

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc tcc 432

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg aat 480

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac gcc 528

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc aag 576

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc gac 624

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc ctg 672

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

agc tcc ccc gtc acc aag agc ttc aac agg ggg gag tgt 711

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 67

<211> 237

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 67

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val

20 25 30

Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly

50 55 60

Gln Gln Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly

65 70 75 80

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

85 90 95

Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys Gln

100 105 110

Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

130 135 140

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

145 150 155 160

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

165 170 175

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

195 200 205

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

210 215 220

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 68

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T5L

<220>

<221> CDS

<222> (1)..(714)

<400> 68

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg ctt acc cag agc cct gac tcc ctt gcc 96

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

gtg tcc ctg ggc gag aga gcc acc atc aac tgt aga gcc tcc cag agc 144

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

ggc cag cct cct aag ctt ctg atc tac aga gcc tcc aac ctt gcc agc 240

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc atc cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc 288

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg acc atc agc tcc ctt cag gcc gag gac gtg gcc acc tac tac tgt 336

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Tyr Cys

100 105 110

cag cag agc aga aag tcc cct tgg acc ttc ggc cag ggc acc aag gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 69

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 69

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 70

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-T6L

<220>

<221> CDS

<222> (1)..(714)

<400> 70

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gac atc gtg ctt acc cag agc cct gac tcc ctt gcc 96

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

gtg tcc ctg ggc gag aga gcc acc atc agc tgt aga gcc tcc cag agc 144

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc atc tcc ggc tac agc ttc atc cac tgg tac cag cag aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

ggc cag cct cct aga ctg ctg atc tac aga gcc tcc aac ctt gcc agc 240

Gly Gln Pro Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc atc cct gcc aga ttc tcc ggc agc ggc tcc ggc acc gac ttc acc 288

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg acc atc agc tcc ctt cag gcc gag gac gtg gcc acc tac ttc tgt 336

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys

100 105 110

cag cag agc aga aag tcc cct tgg acc ttc gcc ggc ggc acc aag gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 71

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 71

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Thr Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 72

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T7H

<400> 72

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 73

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T8H

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 74

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T9H

<400> 74

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 75

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T10H

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 76

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T11H

<400> 76

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 77

<211> 451

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T12H

<400> 77

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asn Asp Tyr

20 25 30

Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala Glu

50 55 60

Ser Leu Glu Gly Arg Val Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 78

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T7L

<400> 78

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 79

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T8L

<400> 79

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 80

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T9L

<400> 80

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 81

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T10L

<400> 81

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 82

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T11L

<400> 82

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 83

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T12L

<400> 83

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 84

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T13L

<400> 84

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 85

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T14L

<400> 85

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 86

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T15L

<400> 86

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 87

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T16L

<400> 87

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 88

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T17L

<400> 88

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 89

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T18L

<400> 89

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 90

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T19L

<400> 90

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 91

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T20L

<400> 91

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 92

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T21L

<400> 92

Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 93

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T22L

<400> 93

Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 94

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T23L

<400> 94

Glu Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 95

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T24L

<400> 95

Glu Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 96

<211> 218

<212> PRT

<213> Artificial sequence

<220>

<223> h#32A1-T25L

<400> 96

Asp Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ile Ser

20 25 30

Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Ser Pro Trp Thr Phe Ala Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 97

<211> 14

<212> PRT

<213> Rattus norvegicus

<400> 97

Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe Tyr Ala

1 5 10

<210> 98

<211> 1398

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-H1-1

<220>

<221> CDS

<222> (1)..(1398)

<400> 98

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gaa gtc cag ctt gtg gaa agc gga ggg gga ctc gtt cag 96

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cca gga ggc tct ctg cgc ctg tca tgc gct gcc agc gga ttt aat ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

aat gat tat ttt atg aac tgg gtc agg cag gct ccg gga aaa ggg ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

gaa tgg gtc gcc cag atc aga aac aag atc tat act tac gct aca ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gca tct gta aag ggg agg ttt aca att agt cgc gac aat gca 288

Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

aaa aat agt ctg tat ctc caa atg aac tcc ctc cgc gca gag gat act 336

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

gct gtc tac tac tgc gcc agg tcc ttg act ggc ggc gac tat ttt gat 384

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg gga cag ggc acc ctg gtg acg gtg agc tca gcc agc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cct tcc gtg ttc cct ctg gcc cct tgt agc cgt tcc acc agc gag 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

tcc acc gcc gcc ctt ggc tgt ctg gtg aag gac tac ttc cct gag cct 528

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tcc gga gcc ctt acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc cct gcc gtg ctg cag tcc agc ggc ctt tac tcc ctg agc tcc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg cct agc tcc aac ttc ggc acc caa acc tac acc tgt aac 672

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

gtg gac cac aag cct agc aac acc aag gtg gac aag acc gtg gag cgt 720

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

aag tgt tgt gtg gag tgt cct cct tgt cct gcc cct cct gtg gcc gga 768

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

cct tcc gtg ttc ctt ttc cct cct aag cct aag gac acc ctg atg atc 816

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

agc cgt acc cct gag gtg acc tgt gtg gtg gtg gac gtg tcc cac gag 864

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

gac cct gag gtg cag ttc aac tgg tac gtg gac ggc gtg gag gtg cac 912

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

aac gcc aag acc aag cct cgt gag gag caa ttc aac agc acc ttc cgt 960

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

gtg gtg tcc gtg ctt acc gtg gtg cac caa gac tgg ctg aac ggc aag 1008

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

gag tac aag tgt aag gtg agc aac aag gga ctt cct gcc cct atc gag 1056

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

aag acc atc tcc aag acc aag ggc caa cct cgt gag cct caa gtg tac 1104

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

acc ctt cct cct agc cgt gag gag atg acc aag aac caa gtg tcc ctt 1152

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

acc tgt ctg gtg aag ggc ttc tac cct agc gac atc gcc gtg gag tgg 1200

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

gag tcc aac gga caa cct gag aac aac tac aag acc acc cct cct atg 1248

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

ctt gac agc gac ggc tcc ttc ttc ctg tac agc aag ctg acc gtg gac 1296

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

aag tcc cgt tgg caa caa ggc aac gtg ttc agc tgt tcc gtg atg cac 1344

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

gag gcc ctg cac aac cac tac acc caa aag agc ctt tcc ctg agc cct 1392

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gga aag 1398

Gly Lys

465

<210> 99

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 99

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 100

<211> 1398

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-H5

<220>

<221> CDS

<222> (1)..(1398)

<400> 100

atg aaa cac ctg tgg ttc ttc ctc ctg ctg gtg gca gct ccc aga tgg 48

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

gtg ctg agc gag gtg cag gtg gtg gag agc ggc ggc gga ctt gtg cag 96

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

cct ggc ggc tcc ctg aga ctt agc tgt gcc acc tcc ggc ttc aac ttc 144

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

aac gac tac ttc atg aac tgg gtg aga cag gcc cct ggc aag ggc ctg 192

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

gag tgg gtg gcc cag atc aga aac aag atc tac acc tac gcc acc ttc 240

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

tac gcc gag agc ctt gag ggc aga ttc acc atc tcc aga gac aac agc 288

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

aag tcc acc gtg tac ctt cag atg aac tcc ctg aga gcc gag gac acc 336

Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

gcc gtg tac tac tgt acc aga agc ctt acc ggc ggc gac tac ttc gac 384

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

tac tgg ggc cag ggc acc ctg gtg acc gtg agc tca gcc agc acc aag 432

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

ggc cct tcc gtg ttc cct ctg gcc cct tgt agc cgt tcc acc agc gag 480

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

tcc acc gcc gcc ctt ggc tgt ctg gtg aag gac tac ttc cct gag cct 528

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

gtg acc gtg agc tgg aac tcc gga gcc ctt acc agc ggc gtg cac acc 576

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

ttc cct gcc gtg ctg cag tcc agc ggc ctt tac tcc ctg agc tcc gtg 624

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

gtg acc gtg cct agc tcc aac ttc ggc acc caa acc tac acc tgt aac 672

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

gtg gac cac aag cct agc aac acc aag gtg gac aag acc gtg gag cgt 720

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

aag tgt tgt gtg gag tgt cct cct tgt cct gcc cct cct gtg gcc gga 768

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

cct tcc gtg ttc ctt ttc cct cct aag cct aag gac acc ctg atg atc 816

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

agc cgt acc cct gag gtg acc tgt gtg gtg gtg gac gtg tcc cac gag 864

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

gac cct gag gtg cag ttc aac tgg tac gtg gac ggc gtg gag gtg cac 912

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

aac gcc aag acc aag cct cgt gag gag caa ttc aac agc acc ttc cgt 960

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

gtg gtg tcc gtg ctt acc gtg gtg cac caa gac tgg ctg aac ggc aag 1008

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

gag tac aag tgt aag gtg agc aac aag gga ctt cct gcc cct atc gag 1056

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

aag acc atc tcc aag acc aag ggc caa cct cgt gag cct caa gtg tac 1104

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

acc ctt cct cct agc cgt gag gag atg acc aag aac caa gtg tcc ctt 1152

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

acc tgt ctg gtg aag ggc ttc tac cct agc gac atc gcc gtg gag tgg 1200

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

gag tcc aac gga caa cct gag aac aac tac aag acc acc cct cct atg 1248

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

ctt gac agc gac ggc tcc ttc ttc ctg tac agc aag ctg acc gtg gac 1296

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

aag tcc cgt tgg caa caa ggc aac gtg ttc agc tgt tcc gtg atg cac 1344

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

gag gcc ctg cac aac cac tac acc caa aag agc ctt tcc ctg agc cct 1392

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gga aag 1398

Gly Lys

465

<210> 101

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 101

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp

1 5 10 15

Val Leu Ser Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Phe

35 40 45

Asn Asp Tyr Phe Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Gln Ile Arg Asn Lys Ile Tyr Thr Tyr Ala Thr Phe

65 70 75 80

Tyr Ala Glu Ser Leu Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

85 90 95

Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Thr Arg Ser Leu Thr Gly Gly Asp Tyr Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

130 135 140

Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

145 150 155 160

Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val

195 200 205

Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn

210 215 220

Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg

225 230 235 240

Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 102

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-L2-15

<220>

<221> CDS

<222> (1)..(714)

<400> 102

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gaa att ctg atg acg cag agt cct gca act ctt agt 96

Gly Ala Tyr Gly Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

ctg tca cct ggc gag aga gcc aca ctc agc tgc cga gcg tcc cag tcc 144

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

gtg acc att agc ggc tat tct ttt att cat tgg tat cag caa aag cct 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

gga cag gcg cca agg ctg ctc att tac aga gca agc aac ctt gcc tct 240

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc att cca gca aga ttc agc ggg agc gga tca ggg aca gat ttc acc 288

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ttg acc atc tcc tcc ctg gag ccg gag gat ttc gcg ttg tat tat tgt 336

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Tyr Cys

100 105 110

cag caa tct agg aag agt cca tgg aca ttt ggc cag ggc acc aaa gtg 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

gag atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 103

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 103

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Tyr Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 104

<211> 714

<212>DNA

<213> Artificial sequence

<220>

<223> h#32A1-L2-16

<220>

<221> CDS

<222> (1)..(714)

<400> 104

atg gtg ctg cag acc cag gtg ttc atc tcc ctg ctg ctg tgg atc tcc 48

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

ggc gca tat ggc gaa atc ctg atg aca cag agt cct gcg acc ctc tcc 96

Gly Ala Tyr Gly Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

ctc tca cca ggc gag agg gcc acc ctg tct tgt aga gcc agc cag tca 144

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

gtc act att agt gga tac tca ttt atc cat tgg tat caa cag aaa cca 192

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

gga cag gcg cct cgg ctt ctg atc tac cgc gcc tca aac ctt gcc tct 240

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

ggc atc ccc gcg agg ttc tct ggc tct ggc agc ggt acc gat ttt aca 288

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

ctg act atc tca agc ctc gaa cct gaa gac ttc gca ctg tac ttt tgc 336

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Phe Cys

100 105 110

cag cag agc agg aag tcc ccc tgg act ttt gca cag gga acc aaa gtc 384

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gln Gly Thr Lys Val

115 120 125

gaa atc aag cgt acg gtg gcc gcc ccc tcc gtg ttc atc ttc ccc ccc 432

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

tcc gac gag cag ctg aag tcc ggc acc gcc tcc gtg gtg tgc ctg ctg 480

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

aat aac ttc tac ccc aga gag gcc aag gtg cag tgg aag gtg gac aac 528

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

gcc ctg cag tcc ggg aac tcc cag gag agc gtg acc gag cag gac agc 576

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

aag gac agc acc tac agc ctg agc agc acc ctg acc ctg agc aaa gcc 624

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

gac tac gag aag cac aag gtg tac gcc tgc gag gtg acc cac cag ggc 672

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

ctg agc tcc ccc gtc acc aag ag agc ttc aac agg ggg gag tgt 714

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 105

<211> 238

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 105

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Thr Ile Ser Gly Tyr Ser Phe Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Arg Ala Ser Asn Leu Ala Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Leu Tyr Phe Cys

100 105 110

Gln Gln Ser Arg Lys Ser Pro Trp Thr Phe Ala Gln Gly Thr Lys Val

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

145 150 155 160

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

165 170 175

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

180 185 190

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly

210 215 220

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 106

<211> 1111

<212>DNA

<213> Artificial sequence

<220>

<223> Nucleic acid sequence encoding signal peptide and constant region of human IgG2 heavy chain

<400> 106

aagcttgcta gcgccaccat gaaacacctg tggttcttcc tcctgctggt ggcagctccc 60

agatgggtgc tgagccaggt gcaattgtgc aggcggttag ctcagccagc accaagggcc 120

cttccgtgtt ccctctggcc ccttgtagcc gttccaccag cgagtccacc gccgcccttg 180

gctgtctggt gaaggactac ttccctgagc ctgtgaccgt gagctggaac tccggagccc 240

ttaccagcgg cgtgcacacc ttccctgccg tgctgcagtc cagcggcctt tactccctga 300

gctccgtggt gaccgtgcct agctccaact tcggcaccca aacctacacc tgtaacgtgg 360

accacaagcc tagcaacacc aaggtggaca agaccgtgga gcgtaagtgt tgtgtggagt 420

gtcctccttg tcctgcccct cctgtggccg gaccttccgt gttccttttc cctcctaagc 480

ctaaggacac cctgatgatc agccgtaccc ctgaggtgac ctgtgtggtg gtggacgtgt 540

cccacgagga ccctgaggtg cagttcaact ggtacgtgga cggcgtggag gtgcacaacg 600

ccaagaccaa gcctcgtgag gagcaattca acagcacctt ccgtgtggtg tccgtgctta 660

ccgtggtgca ccaagactgg ctgaacggca aggagtacaa gtgtaaggtg agcaacaagg 720

gacttcctgc ccctatcgag aagaccatct ccaagaccaa gggccaacct cgtgagcctc 780

aagtgtacac ccttcctcct agccgtgagg agatgaccaa gaaccaagtg tcccttacct 840

gtctggtgaa gggcttctac cctagcgaca tcgccgtgga gtgggagtcc aacggacaac 900

ctgagaacaa ctacaagacc accccctccta tgcttgacag cgacggctcc ttcttcctgt 960

acagcaagct gaccgtggac aagtcccgtt ggcaacaagg caacgtgttc agctgttccg 1020

tgatgcacga ggccctgcac aacccactaca cccaaaagag cctttccctg agccctggaa 1080

agtgatatcg ggcccgttta aacgggtggc a 1111

Claims (61)

1. the antigen-binding fragment of a kind of antibody or the antibody, the antibody binding is by SEQ ID NO:2 amino acid represented The polypeptide of the amino acid residue 39-165 compositions of sequence, and suppress osteoclast formation and/or osteoclastic property bone information, the antibody Antigen-binding fragment be selected from Fab, F (ab ') 2, Fab ' and Fv,

It is characterized in that contain：By SEQ ID NO:The weight of the amino acid residue 20-140 compositions of 41 amino acid sequences represented Chain variable region sequence and by SEQ ID NO:The light chain variable of the amino acid residue 21-132 compositions of 43 amino acid sequences represented Region sequence.

2. the antigen-binding fragment of antibody according to claim 1 or the antibody, it is characterised in that it is scFv.

3. the antigen-binding fragment of antibody according to claim 1 or the antibody, it is characterised in that the antibody is embedding Close antibody.

4. the antigen-binding fragment of antibody according to claim 3 or the antibody, it is characterised in that by sequence of heavy chain and Sequence of light chain forms, and the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 groups of 41 amino acid sequences represented Into, and the sequence of light chain is by SEQ ID NO:The amino acid residue 21-237 compositions of 43 amino acid sequences represented.

5. the antigen-binding fragment of antibody according to claim 1 or the antibody, it is characterised in that the antibody is people Source.

6. the antigen-binding fragment of antibody according to claim 4 or the antibody, wherein there is the heavy chain people to be immunized The constant region of the heavy chain of Lysozyme 2, and the light chain has the constant region of human immunoglobulin(HIg) κ light chains.

7. a kind of suppression osteoclast formation and/or the antibody of osteoclastic property bone information or the antigen-binding fragment of the antibody, its Described in antibody contain：

(a) it is selected from the weight chain variable district of following amino acid sequences：

A1 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 51 amino acid sequences represented Into；

A2 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 53 amino acid sequences represented Into；

A3 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 55 amino acid sequences represented Into；

A4 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 57 amino acid sequences represented Into；

A5 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 59 amino acid sequences represented Into；

A6 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 99 amino acid sequences represented Into；

A7 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 20-140 groups of 101 amino acid sequences represented Into；

A8 a kind of) amino acid sequence, its with selected from a1) to a7) and any one amino acid sequence there is at least 95% homology；With

A9 a kind of) amino acid sequence, it is included in and is selected from a1) to a7) any one amino acid sequence in an amino acid it is residual Displacement, deletion or the addition of base；With

(b) it is selected from the light chain variable district of following amino acid sequences：

B1 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 61 amino acid sequences represented Into；

B2 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 63 amino acid sequences represented Into；

B3 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 65 amino acid sequences represented Into；

B4 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-132 groups of 67 amino acid sequences represented Into；

B5 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 69 amino acid sequences represented Into；

B6 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 71 amino acid sequences represented Into；

B7 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 103 amino acid sequences represented Into；

B8 a kind of) amino acid sequence, it is by SEQ ID NO:The amino acid residue 21-133 groups of 105 amino acid sequences represented Into；

B9 a kind of) amino acid sequence, its with selected from b1) to b8) and any one amino acid sequence there is at least 95% homology；With

B10 a kind of) amino acid sequence, it is included in and is selected from b1) to b8) any one amino acid sequence in an amino acid Displacement, deletion or the addition of residue,

Wherein described weight chain variable district has CDRH1, CDRH2 and CDRH3, and the CDRH1 is by SEQ ID NO:44 ammonia represented Base acid sequence forms, and the CDRH2 is by SEQ ID NO:45 and SEQ ID NO:97 any one amino acid sequence group represented Into, and the CDRH3 is by SEQ ID NO:The 46 amino acid sequence compositions represented；And

The light chain variable district has CDRL1, CDRL2 and CDRL3, and the CDRL1 is by SEQ ID NO:47 amino acid represented Sequence forms, and the CDRL2 is by SEQ ID NO:The 48 amino acid sequence compositions represented, and the CDRL3 is by SEQ ID NO: The 49 amino acid sequence compositions represented；And

Wherein described antigen-binding fragment is selected from Fab, F (ab ') 2, Fab ' and Fv.

8. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain：

(a) with the a1 selected from claim 7) to a7) and any one amino acid sequence there is the amino acid of at least 99% homology Sequence；With

(b) with the b1 selected from claim 7) to b8) and any one amino acid sequence there is the amino acid of at least 99% homology Sequence.

9. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 51 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:61 amino acid sequences represented Amino acid residue 21-133 composition.

10. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 53 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:63 amino acid sequences represented Amino acid residue 21-133 composition.

11. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 55 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:65 amino acid sequences represented Amino acid residue 21-133 composition.

12. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 55 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:67 amino acid sequences represented Amino acid residue 21-132 composition.

13. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 57 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:69 amino acid sequences represented Amino acid residue 21-133 composition.

14. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 59 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:71 amino acid sequences represented Amino acid residue 21-133 composition.

15. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 99 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:69 amino acid sequences represented Amino acid residue 21-133 composition.

16. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 101 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:69 amino acid sequences represented Amino acid residue 21-133 composition.

17. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 99 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:The 103 amino acid sequences represented The amino acid residue 21-133 compositions of row.

18. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain contain weight chain variable district, the weight chain variable district is made up of such amino acid sequence, its By SEQ ID NO:The amino acid residue 20-140 compositions of 99 amino acid sequences represented, and the sequence of light chain contains light chain Variable region, the light chain variable district are made up of such amino acid sequence, and it is by SEQ ID NO:The 105 amino acid sequences represented The amino acid residue 21-133 compositions of row.

19. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 51 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 61 amino acid sequences represented.

20. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 53 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 63 amino acid sequences represented.

21. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 55 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 65 amino acid sequences represented.

22. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 55 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-237 compositions of 67 amino acid sequences represented.

23. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 57 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 69 amino acid sequences represented.

24. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-470 compositions of 59 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 71 amino acid sequences represented.

25. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-466 compositions of 99 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 69 amino acid sequences represented.

26. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-466 groups of 101 amino acid sequences represented Into, and the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 69 amino acid sequences represented.

27. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-466 compositions of 99 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 103 amino acid sequences represented.

28. the antigen-binding fragment of antibody according to claim 7 or the antibody, it is characterised in that contain sequence of heavy chain And sequence of light chain, the sequence of heavy chain is by SEQ ID NO:The amino acid residue 20-466 compositions of 99 amino acid sequences represented, And the sequence of light chain is by SEQ ID NO:The amino acid residue 21-238 compositions of 105 amino acid sequences represented.

29. a kind of pharmaceutical composition, it is characterised in that include at least one antibody according to claim 1-28 or described The antigen-binding fragment of antibody.

30. pharmaceutical composition according to claim 29, it is characterised in that be therapeutic agent and/or the prevention of abnormal bone metabolism Agent.

A kind of 31. use of the antigen-binding fragment comprising at least one antibody according to claim 1-28 or the antibody In the pharmaceutical composition for treating and/or preventing abnormal bone metabolism, wherein described pharmaceutical composition further includes at least one choosing From following members：Diphosphonate, activated vitamin D₃, calcitonin, estradiol, SERM (selective estrogen receptor adjust Agent), Ipriflavone, vitamin K₂(Menatetrenone), calcium preparation, PTH (parathyroid hormone), non-steroid anti-inflammatory drug, (parathyroid hormone is related by soluble TNF acceptor, the antigen-binding fragment of anti-TNF-Alpha antibodies or the antibody, anti-PTHrP Albumen) antibody or the antibody antigen-binding fragment, IL-1 receptor antagonists, anti-IL-6 receptor antibodies or the antibody Antigen-binding fragment, (osteoclast generation suppresses for the antigen-binding fragment and OCIF of anti-RANKL antibody or the antibody The factor).

32. pharmaceutical composition according to claim 30, wherein the abnormal bone metabolism is selected from：It is osteoporosis, adjoint The osteoclasia of rheumatoid arthritis, carcinous hypercalcinemia, with the osteoclasia of Huppert's disease or the Bone tumour of cancer, big and small Born of the same parents' knurl, sclerotin reduce, anodontia caused by periodontitis, the bone dissolving around joint prosthesis, the osteoclasia in chronic osteomyelitis, bone Paget disease, renal osteodystrophy and osteogenesis imperfecta.

33. pharmaceutical composition according to claim 32, it is characterised in that the abnormal bone metabolism is osteoporosis, companion The osteoclasia of osteoclasia with rheumatoid arthritis or the Bone tumour with cancer.

34. pharmaceutical composition according to claim 33, it is characterised in that the abnormal bone metabolism is osteoporosis.

35. pharmaceutical composition according to claim 34, it is characterised in that the osteoporosis is that post menopausal sclerotin is dredged Loose disease, senile osteoporosis, the secondary osteoporosis as caused by using therapeutic agent or adjoint rheumatoid arthritis Osteoporosis.

36. pharmaceutical composition according to claim 35, it is characterised in that the therapeutic agent is selected from steroids and immune suppression Preparation.

37. pharmaceutical composition according to claim 31, wherein the abnormal bone metabolism is selected from：It is osteoporosis, adjoint The osteoclasia of rheumatoid arthritis, carcinous hypercalcinemia, with the osteoclasia of Huppert's disease or the Bone tumour of cancer, big and small Born of the same parents' knurl, sclerotin reduce, anodontia caused by periodontitis, the bone dissolving around joint prosthesis, the osteoclasia in chronic osteomyelitis, bone Paget disease, renal osteodystrophy and osteogenesis imperfecta.

38. the pharmaceutical composition according to claim 37, it is characterised in that the abnormal bone metabolism is osteoporosis, companion The osteoclasia of osteoclasia with rheumatoid arthritis or the Bone tumour with cancer.

39. the pharmaceutical composition according to claim 38, it is characterised in that the abnormal bone metabolism is osteoporosis.

40. the pharmaceutical composition according to claim 39, it is characterised in that the osteoporosis is that post menopausal sclerotin is dredged Loose disease, senile osteoporosis, the secondary osteoporosis as caused by using therapeutic agent or adjoint rheumatoid arthritis Osteoporosis.

41. pharmaceutical composition according to claim 40, it is characterised in that the therapeutic agent is selected from steroids and immune suppression Preparation.

42. the application for being used for medicine production of the antibody or its antigen-binding fragment according to claim 1-28, the medicine Thing is used to treat and/or prevent abnormal bone metabolism.

43. the application for being used for medicine production of the antibody or its antigen-binding fragment according to claim 1-28, the medicine Thing is used to treat and/or prevent abnormal bone metabolism, it is characterised in that simultaneously or sequentially using described antibody or antigen binding fragment Section is selected from following members with least one：Diphosphonate, activated vitamin D₃, calcitonin, estradiol, SERM (selectivity it is female Hormone receptor modulator), Ipriflavone, vitamin K₂(Menatetrenone), calcium preparation, PTH (parathyroid hormone), non-steroid Body AID, soluble TNF acceptor, antigen-binding fragment, anti-PTHrP (the first shapes of anti-TNF-Alpha antibodies or the antibody Other glandular hormone GAP-associated protein GAP) antigen-binding fragment, IL-1 receptor antagonists, the anti-IL-6 acceptors of antibody or the antibody resist The antigen-binding fragment and OCIF of the antigen-binding fragment of body or the antibody, anti-RANKL antibody or the antibody are (osteoclastic Hemapoiesis inhibiting factor).

44. application according to claim 42, it is characterised in that the abnormal bone metabolism is osteoporosis, with class wind The osteoclasia of the osteoclasia of wet arthritis or the Bone tumour of adjoint cancer.

45. application according to claim 44, it is characterised in that the abnormal bone metabolism is osteoporosis.

46. application according to claim 45, it is characterised in that the osteoporosis is PMO, old Year property osteoporosis, the secondary osteoporosis as caused by using therapeutic agent are dredged with the sclerotin of rheumatoid arthritis Loose disease.

47. application according to claim 46, it is characterised in that the therapeutic agent is selected from steroids and immunodepressant.

48. application according to claim 43, it is characterised in that the abnormal bone metabolism is osteoporosis, with class wind The osteoclasia of the osteoclasia of wet arthritis or the Bone tumour of adjoint cancer.

49. application according to claim 48, it is characterised in that the abnormal bone metabolism is osteoporosis.

50. application according to claim 49, it is characterised in that the osteoporosis is PMO, old Year property osteoporosis, the secondary osteoporosis as caused by using therapeutic agent are dredged with the sclerotin of rheumatoid arthritis Loose disease.

51. application according to claim 50, it is characterised in that the therapeutic agent is selected from steroids and immunodepressant.

52. a kind of polynucleotides, it encodes the antibody according to any one of claim 1,4 and 7-28.

53. polynucleotides according to claim 52, it is characterised in that contain：By SEQ ID NO:40 nucleosides represented The nucleotide sequence of the nucleotides 58-420 compositions of acid sequence, and by SEQ ID NO:The nucleosides of 42 nucleotide sequences represented The nucleotide sequence of sour 61-396 compositions.

54. polynucleotides according to claim 53, it is characterised in that contain：By SEQ ID NO:40 nucleosides represented The nucleotide sequence of the nucleotides 58-1410 compositions of acid sequence, and by SEQ ID NO:The nucleosides of 42 nucleotide sequences represented The nucleotide sequence of sour 61-711 compositions.

55. polynucleotides according to claim 52, it is characterised in that contain：

(a) a kind of polynucleotides, it is selected from following nucleotide sequences：

A1 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 50 nucleotide sequences represented；

A2 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 52 nucleotide sequences represented；

A3 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 54 nucleotide sequences represented；

A4 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 56 nucleotide sequences represented；

A5 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 58 nucleotide sequences represented；

A6 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 98 nucleotide sequences represented；

A7 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 58-420 compositions of 100 nucleotide sequences represented；

A8) the nucleotide sequence of polynucleotides, the polynucleotides under strict conditions with by selected from a1) to a7) and any one The polynucleotides hybridization of the complementary nucleotide sequence composition of nucleotide sequence；With

A9 a kind of) nucleotide sequence, it is included in and is selected from a1) to a7) any one nucleotide sequence in a nucleotides Displacement, delete or add；With

(b) a kind of polynucleotides, it is selected from following nucleotide sequences：

B1 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 60 nucleotide sequences represented；

B2 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 62 nucleotide sequences represented；

B3 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 64 nucleotide sequences represented；

B4 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-396 compositions of 66 nucleotide sequences represented；

B5 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 68 nucleotide sequences represented；

B6 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 70 nucleotide sequences represented；

B7 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 102 nucleotide sequences represented；

B8 a kind of) nucleotide sequence, it is by SEQ ID NO:The nucleotides 61-399 compositions of 104 nucleotide sequences represented；

B9) the nucleotide sequence of polynucleotides, the polynucleotides under strict conditions with by selected from b1) to b8) and any one The polynucleotides hybridization of the complementary nucleotide sequence composition of nucleotide sequence；With

B10 a kind of) nucleotide sequence, it is included in and is selected from b1) to b8) any one nucleotide sequence in a nucleotides Displacement, deletion or addition.

56. polynucleotides according to claim 55, it is characterised in that containing such polynucleotides, it is included：

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 50 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 60 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 52 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 62 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 54 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 64 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 54 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-396 compositions of 66 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 56 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 68 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 58 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 70 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 68 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 100 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 compositions of 68 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 groups of 102 nucleotide sequences represented Into；Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-420 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-399 groups of 104 nucleotide sequences represented Into；Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 50 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 60 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 52 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 62 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 54 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 64 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 54 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-711 compositions of 66 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 56 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 68 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1410 compositions of 58 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 70 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1398 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 compositions of 68 nucleotide sequences represented； Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1398 compositions of 100 nucleotide sequences represented；With by so Nucleotide sequence composition polynucleotides, it is by SEQ ID NO:The nucleotides 61-714 groups of 68 nucleotide sequences represented Into；Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1398 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 groups of 102 nucleotide sequences represented Into；Or

By SEQ ID NO:The nucleotide sequence of the nucleotides 58-1398 compositions of 98 nucleotide sequences represented；With by such The polynucleotides of nucleotide sequence composition, it is by SEQ ID NO:The nucleotides 61-714 groups of 104 nucleotide sequences represented Into.

57. a kind of carrier, it includes the polynucleotides according to claim any one of 52-56.

58. a kind of host cell of conversion, it includes the polynucleotides according to claim any one of 52-56.

59. a kind of host cell of conversion, it includes carrier according to claim 57.

A kind of 60. antigen binding fragment of antibody produced according to any one of claim 1,4 and 7-28 or the antibody The method of section, methods described include：Host cell according to claim 58 is cultivated, and is purified from obtained cultured products Antibody.

A kind of 61. antigen binding fragment of antibody produced according to any one of claim 1,4 and 7-28 or the antibody The method of section, methods described include：Host cell according to claim 59 is cultivated, and is purified from obtained cultured products Antibody.